Biological Conservation 286 (2023) 110268 Contents lists available at ScienceDirect Biological Conservation journal homepage: www.elsevier.com/locate/biocon Reappraising the use of forearm rings for bat species Lourdes Lobato-Bailón a, *, Adrià López-Baucells b, David Guixé c, Carles Flaquer b, Jordi Camprodon c, d, Xavier Florensa-Rius c, Maria Mas b, Laura Torrent b, c, Laura Ordeix e, Oriol Tallo-Parra f, Maria P. Ribas a, Ignasi Marco a, Annaïs Carvajal g, Manel López-Bejar g, Sebastian Napp h, i, Lola Pailler-García h, i, Johan Espunyes a, Oscar Cabezón a, i a Wildlife Conservation Medicine Research Group (WildCoM), Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Catalonia, Spain b BiBio (Biodiversity and Bioindicators Research Group), Natural Sciences Museum of Granollers, 08402 Granollers, Catalonia, Spain c Forest Science and Technology Centre of Catalonia (CTFC), Conservation Biology Group (GBiC), 25280 Solsona, Catalonia, Spain d Departament de Biociències, Universitat de Vic-Universitat Central de Catalunya, 08500 Vic, Catalonia, Spain e Fundació Hospital Clínic Veterinari and Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona (UAB) campus, 08193 Bellaterra, Catalonia, Spain f Animal Welfare Education Centre (AWEC) and Department of Animal and Food Science, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Catalonia, Spain g Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Catalonia, Spain h IRTA Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la UAB, 08193 Bellaterra, Catalonia, Spain i Unitat mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona (UAB) campus, 08193 Bellaterra, Catalonia, Spain A R T I C L E I N F O A B S T R A C T Keywords: Long-term mark-recapture studies are essential for bat conservation. Over the last decades, millions of bats across Bat Europe and America have been marked with forearm rings for this purpose. Although it is considered a cost- Forearm ring effective method compared to Passive Integrated Transponders (PIT) tags, direct injuries from using forearm PIT tag rings have been reported since their very first use. Yet, their impact on bats’ welfare has not been systematically Conservation Animal welfare evaluated and remains a highly controversial issue among the scientific community and policymakers. Here we assess the impact of forearm rings and PIT tags on the health of different bat species. We reviewed 12 years of the existing recapture data of free-ranging bats from NE Spain and evaluated the impact of both marking tools in a captive colony of Carollia perspicillata, by assessing the development of skin lesions and levels of cortisol me­ tabolites in guano (CG) after marking. We report that 55.1 % (435/790) of the recaptured free-ranging bats with forearm rings presented skin lesions. All banded C. perspicillata (n = 22, 100 %) developed skin lesions, whereas none of the PIT-tagged (n = 21) presented lesions. Levels of CG were significantly higher after marking with forearm rings only for one group. Banded C. perspicillata exhibited discomfort-associated behaviours due to forearm rings. Under the “precautionary principle”, we recommend the ban of forearm rings for all bat species until species-specific studies under controlled conditions are performed and approved by a legally constituted ethics committee. Consideration of other long-term marking tools is mandatory to align with global bat con­ servation strategies. 1. Introduction shown downward trends for the past 50 years, especially throughout Western Europe (Hutson et al., 2001). At present, 22 % of the European Bat populations have experienced significant declines over the past bat species are classified as Endangered (EN) or Vulnerable (VU) by the century, leading the International Union for Conservation of Nature IUCN (Hutson et al., 2001; IUCN, 2022) but strict species and habitat (IUCN) to classify nearly half of the species as threatened or near protection, accompanied by investments in research to improve con­ threatened globally (IUCN, 2022). In Europe, bat populations have servation strategies, have stabilized populations for a number of species * Corresponding author. E-mail address: l.lobatobailon@gmail.com (L. Lobato-Bailón). https://doi.org/10.1016/j.biocon.2023.110268 Received 8 June 2023; Received in revised form 9 August 2023; Accepted 29 August 2023 Available online 10 September 2023 0006-3207/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). L. Lobato-Bailón et al. Biological Conservation 286 (2023) 110268 (Browning et al., 2021; Haysom et al., 2013). However, population insectivorous bats from Catalonia (NE Spain) from 12 years (2009 to trends and key threatening processes are not fully understood for the 2021) and 2) monitoring skin injuries, assessing cortisol metabolites in vast majority of species. guano and observing alterations in the behaviour of captive Carollia The Agreement on the Conservation of Populations of European Bats perspicillata, derived from both marking tools, under captive conditions (UNEP/EUROBATS) was set up in 1991 to cooperate towards the con­ in a zoological institution. servation of bats throughout Europe. The Agreement provides guidelines to be implemented by European institutions and conservationists on 2. Materials and methods diverse topics, including the capture and marking of bats (Hutson et al., 2001; UNEP/EUROBATS, 2003). For decades, the capture-mark- 2.1. Assessment of the impact of forearm rings in free-ranging recaptured recapture technique has been used in wildlife for research and conser­ bats from Catalonia, NE Spain vation (Cody and Smallwood, 1996; Gaisler and Chytil, 2002; Shenbrot et al., 2010) Particularly, the recognised challenge of providing infor­ We analysed data from bat recaptures in NE-Spain, from the last 12 mation about bat ecology, population dynamics and social behaviour years (2009–2021), on forearm rings and PIT tags (Table A1, Appendix identifies long-term mark-recapture studies as an important tool for bat A) collected by two institutions for the research of bats in Catalonia conservation (Locatelli et al., 2019). At present, most of these long-term (BiBio Research Group - Natural Sciences Museum of Granollers and bat monitoring studies are undertaken using two marking tools: forearm Centre de Ciència i Tecnologia Forestal de Catalunya). rings (also dubbed bands) and Passive Integrated Transponder (PIT) tags We calculated the proportion of bats detected with injuries from the (Kunz and Parsons, 2009; Mitchell-Jones and McLeish, 2004). Tagging total recaptured per species and marking method, with exact confidence with forearm rings is a cost-effective method for marking bats, compared intervals of 95 %. First, considering all the recaptured bats, we tested to PIT tags, and no technical device is required to read it. These attri­ whether there was a statistically significant difference in the frequency butes have given bands a rising popularity among bat researchers and of lesions between bats marked with forearm rings and bats marked with conservationists (Godinho et al., 2015; van Harten et al., 2019; van PIT tags using the Fisher’s exact test. Then, for recaptured bats marked Harten et al., 2022a). with forearm rings, we also evaluated whether there were statistically Millions of bats have been marked with forearm rings worldwide, significant differences in the frequency of lesions between the different especially in Europe and North America, over the last decades (Ellison, species of bats. To do that, we used pairwise Fisher’s exact tests with the 2008; Hutson et al., 2001; Hutterer et al., 2005). However, direct in­ Holm’s correction to account for multiple comparisons. Only those juries from using bands have been reported, suggesting that this marking species for which there were at least seven individuals were included in technique could silently compromise the conservation efforts of bat the comparison. populations worldwide (Baker et al., 2001; Balmori and Quetglas, 2000; In a small subset of the bats marked with forearm rings (n = 304), Ellison, 2008; Hutterer et al., 2005). This issue has become highly besides the data on the species, there was supplementary information controversial among the scientific community and policymakers. The collected on the age, sex and reproductive state. The effect of age could recommended ring sizes and designs in Europe are based on field not be evaluated because all animals included in the study were adults. recapture experience from Bulgaria, Germany, The Netherlands, To evaluate whether the other factors had a significant effect on the Portugal, Ukraine, and the United Kingdom (UNEP/EUROBATS, 2003). probability of developing a lesion, a logistic regression model was built However, the UNEP/EUROBATS Agreement recognises that banding is with the variables species, sex, and reproductive state. an invasive procedure and that the degree of tolerance to this marking tool varies among species and populations of the same species (UNEP/ 2.2. Assessment of the impact of tagging microbats in captivity EUROBATS, 2018). For this reason, some countries (i.e. USA, Australia and France) have implemented moratoriums on the use of forearm rings We tested the impact of forearm rings and PIT tags on a captive in bat research based on field studies that reported direct physical in­ colony of adult Seba’s short-tailed bats (Carollia perspicillata), housed at juries in bat wings produced by this marking tool (Baker et al., 2001; Butterfly Park (license number G25-00119), a small zoo established in Ellison, 2008; Hutterer et al., 2005). Currently, the knowledge of the Empuriabrava (NE Spain). These animals were already required to be impact of marking tools on bats’ health is mainly based on field studies individually identified for the zoo collection register. All bats were born with low recapture rates (Ellison, 2008; Hutterer et al., 2005; Steffens in captivity and adapted to the routine institutional management stra­ et al., 2007). As a consequence, the direct effects and the magnitude of tegies: daily cleansing of the facilities between 9:00 and 10:00 and food the impact might be largely underestimated. Furthermore, few studies supply between 18:00 and 19:00 h. have examined the impact of marking tools on survival rates and sub­ The Seba’s short-tailed bat is a widespread Phyllostomidae bat species lethal effects (e.g. reduction in body condition) in free-ranging bats inhabiting Central and South America. This species is classified as Least (Baker et al., 2001; Locatelli et al., 2019; Mellado et al., 2022). These Concern by the IUCN Red List (Barquez et al., 2015) and is exhibited in field studies, although valuable, are unable to explore other collateral many zoological institutions due to its small size, diet specifications effects such as alterations in social or reproductive behaviour, or (mainly frugivorous species) and its gregarious behaviour, roosting in increased stress and susceptibility to disease. groups up to hundreds of individuals (Rasweiler IV et al., 2009). Roosts Measurement of cortisol metabolites in faeces has been broadly used can be organised as a harem (1 adult male with large aggregations of to evaluate stress in animals, including bats (Hernández-Arciga et al., females) and as a bachelor (large aggregations of adult and subadult 2020; Kelm et al., 2016). Sustained high cortisol concentrations have males without females) (Martínez-Medina et al., 2018). The phenotypic been associated with poor body condition, reduced immunocompetence, characteristics of this species (similar in size and morphology to several decreased reproductive success, and ultimately fitness disruption (Cain species of European bats) and its adaptation to a captive environment and Cidlowski, 2017; Edwards et al., 2019). Research on bat marking (Rasweiler IV et al., 2009) made it suitable for the purpose of this study. tools under controlled conditions to document and understand these An intentional sampling method was used to select 32 adult females acute and long-term effects on the health and survival of these species is and 11 adult males from the captive colony of Seba’s short-tailed bats (n crucial. Insights from such controlled studies can help to develop = 150), excluding individuals with scars, solved fractures or any other evidence-based national and international policies to guarantee animal integument abnormalities. Bats were captured with a telescopic net bag welfare and support current bat conservation strategies. (BTHK Tree-Roost net bag, NHBS). Best practices for the care and The aim of the present study was to assess the impact of forearm rings handling of bats (H. Miller, 2016) were followed throughout the study and Passive Integrated Transponder (PIT) tags on the health of a selected and at least one European Diplomate in Veterinary Medicine (Wildlife group of bat species by 1) reviewing the existing recapture data of wild Population Health and/or Veterinary Dermatology) and a bat specialist 2 L. Lobato-Bailón et al. Biological Conservation 286 (2023) 110268 were present during all procedures. Bats were distributed in four groups: of the epidermis. Topical treatment may improve and boost the patient’s group 1 (one male and eight females) and group 2 (one male and 12 recovery time assigned to this stage. Stage 3 included injuries with females) were marked with forearm rings, and group 3 (one male and 12 significant skin tissue and fluid loss, exposure of the dermis and subcutis, females) and group 4 (8 males) were fitted with PIT tags. Each group was and epidermal ischemia. Topical, parenteral, and surgical treatment tested independently and housed at different times in a separate may be necessary for some presentations of Stage 3 to avoid the devel­ enclosure of 50 square meters and 3 m high, meeting the environmental opment of sepsis or loss of extremity function (Miller et al., 2012). requirements of C. perspicillata (relative humidity of 80 % and average minimum and maximum temperature of 20–26 ◦ C) (Bat Taxonomic 2.2.2. Complementary animal welfare monitoring Advisory Group, 2004; Martínez-Medina et al., 2018). The enclosure All bats were continuously monitored by two infrared video cameras provided a green semi-opened area and an artificial cave for light (Foscam FI9926P®) located at the entrance of the artificial cave of the insulation during daylight hours. Additionally, bats were isolated from experimental facility and in front of the feeding plates, respectively. external stressors such as visitors and educational activities for the Video cameras allowed the live view of the animals and stored a whole duration of the study. The adaptation period for each group continuous recording for delayed observation and assessment of spanned four weeks, after which a marking tool (forearm ring or PIT tag) behavioural indicators of welfare. Furthermore, zoo caretakers reported was implemented and tested for six consecutive weeks. Forearm rings any abnormal conduct detected during feeding and cleansing times. were withdrawn at the end of the sixth week in groups 1 and 2 and then Additionally, to explore the indirect effects of forearm rings and PIT those bats were PIT-tagged and returned to their original enclosure. tags on the welfare of bats, we measured cortisol metabolites in guano Forearm rings used for groups 1 and 2 were distributed by the (CG) from each group, twice a week during the adaptation and marking SECEMU Association (Spanish Association for the Conservation and period. Hormone metabolites were extracted following a methanol Research of Bats, Spain) and approved for their use in Spain. They extraction protocol previously described (Palme et al., 2013; Tallo-Parra contained a unique alpha-numerical code, were omega-shaped, 4.2 mm et al., 2014) (Cortisol analysis, Appendix B). in diameter and made of a light alloy of aluminum and magnesium (model 3X, 0.103 g of weight). Bats were banded (Fig. A1, Appendix A) 2.2.3. Statistical analyses by highly experienced personnel. Fingers were used to gently close the A Fisher’s exact test was applied to compare the frequencies of le­ ring, maintaining their round shape and leading to ˷1 mm wide opening sions between groups marked with forearm rings and PIT tags. between the tips so that it was free to slide up and down the forearm but To evaluate whether the marking tool (forearm ring or PIT tag) prevented finger bones from becoming trapped. influenced the levels of CG from the animals in the different groups, a Bats from groups 3 and 4 were PIT-tagged using a sterilised 10 mm × linear regression model was built considering CG as the response vari­ 1.4 mm transponder of 0.036 g of weight (FrenChip Mini, Avid®, Cali­ able, and the time point (sampling time) and the interaction between fornia, USA), injected subcutaneously in the dorsum so that the tag period (adaptation or marking) and group (1–4) as the covariates. The rested between the shoulder blades, as described previously in the comparison between the estimated marginal means was computed. We literature (van Harten et al., 2020). Next, a drop of surgical glue (3M™ hypothesised that CG might be modulated by the stress response pro­ VetBond™) was applied to the injection site to minimise tag loss, and the duced by being tagged. Therefore, intragroup CG concentrations after animal was restrained for a few seconds until the complete drying of the tagging were expected to be significantly higher than those during the adhesive. adaptation period, particularly in the two groups with forearm rings. All the analyses were performed using R programming (R Core Team, 2.2.1. Physical impact assessment 2022) language and RStudio (RStudio Team, 2022) environment. We All bats were physically examined prior to the study and at the end of use the package lsmeans (Least-Squares Means) for the analyses (Russell the adaptation period to ensure they were physically fit and healthy V, 2016) and ggplot2 (Wickham, 2016) and tidyverse (Wickham et al., before any tag was implanted. Particular attention was given to fur and 2019) for the figures. patagia. The evaluation of skin lesions caused by both marking tools was carried out by a Diplomate specialist from The European College of 3. Results Veterinary Dermatology (ECVD) at the end of the six-week period in all four groups. Cutaneous lesions found after marking with forearm rings 3.1. Physical impact of tagging free-ranging bats with forearm rings were classified in order of increasing severity in Stage 1, Stage 2, and Stage 3 (Table A2, Appendix A). Stage 1 was assigned to any mild lesion Information regarding 1236 recaptured free-ranging bats was ana­ in the sense that the epidermis was not damaged, and no treatment was lysed, 790 of which had forearm rings and 446 had PIT tags (Table 1). required after the ring withdrawal. Stage 2 referred to moderate cuta­ Recaptured bats included the following species: Miniopterus schreibersii, neous lesions associated with a superficial breakdown in the continuity Myotis capaccinii, M. daubentonii, M. myotis, Nyctalus lasiopterus, Table 1 Percentage of injured bats due to forearm rings and PIT tags. Data obtained during the last 12 years (2009–2021), from bats recaptured in Catalonia (NE-Spain). Species Marking tool Bats recaptured Bats with injuries Injuries occurrence % (CI95%) M. schreibersii Forearm ring 445 262 58.9 (54.2–63.5) PIT tag 116 0 0.0 (0.0–3.1) M. capaccinii Forearm ring 57 8 14.0 (6.3–25.8) M. daubentonii Forearm ring 3 1 33.3 (0.8–90.6) M. myotis Forearm ring 7 2 28.6 (3.7–71.0) N. lasiopterus Forearm ring 12 0 0.0 (0.0–26.5) PIT tag 5 0 0.0 (0.0–52.2) N. leisleri Forearm ring 105 56 53.3 (43.3–63.1) PIT tag 79 0 0.0 (0.0–4.6) N. noctula Forearm ring 2 0 0.0 (0.0–84.2) P. pygmaeus Forearm ring 159 106 66.7 (58.8–74.0) PIT tag 246 0 0.0 (0.0–1.5) Total Forearm ring 790 435 55.1 (51.5–58.6) PIT tag 446 0 0.0 (0.0–0.8) 3 L. Lobato-Bailón et al. Biological Conservation 286 (2023) 110268 Fig. 1. Differences between proportions of skin lesions found in free-ranging recaptured bats with forearm rings (pairwise Fisher’s exact tests with Holm’s correction, p-value <0.05). The number of bats detected with skin lesions from the total recaptured is depicted in each species box. N. leisleri, N. noctula and Pipistrellus pygmaeus. A total of 435 recaptured C. perspicillata (n = 15, 68.2 %). The most frequently found lesions were bats (55.1 %, CI95%: 51.5–58.6) showed skin lesions derived from the skin depigmentation and thinning (n = 19, 86.4 %), erythema (n = 16, forearm ring, whereas no skin lesions (0 %, CI95%: 0.0–0.8) were 72.7 %) (Fig. 3A) and skin neovascularisation (n = 13, 59.1 %) (Fig. 3). detected in any of the 446 bats recaptured with PIT tags (Table 1). The The area of skin depigmentation and thinning coincided with the loca­ frequency of lesions was significantly higher in bats marked with fore­ tion of ring displacement along the forearm ring, dorsally and ventrally arm rings compared with bats marked with PIT tags (p-value < 0.001). to the radius and ulna. Erythematous plaques (n = 12, 54.5 %) (Fig. 3B) Statistically significant differences between species are shown in Fig. 1. were visible along the same area of skin depigmentation and thinning, The presence or absence of skin lesions was reported for all recap­ presumably in the most proximal area where the ring could be displaced. tured species, but the classification or description of the lesions was only These erythematous plaques were commonly surrounded by neo­ provided for the following four species. The most reported injury in vascularisation, newly formed capillaries arising from the brachial vein M. schreibersii and N. leisleri was a callus formation (55.2 % and 66.7 % (Fig. 3B). Erosions (Fig. 3C) were present in six bats, mostly on the edges respectively) (Fig. 2A), followed by an ulcerated callus (21.2 % and 14.6 of the propatagium close to the carp and in the same area of the % respectively). Ulcerated callus (Fig. 2B) was the most described lesion erythematous plaques, due to forearm ring rubbing. Four (18.2 %) bats (29.4 %%) in P. pygmaeus, followed by callus formation (20.3 %), ulcers developed severe cutaneous lesions, corresponding with ulcers (Fig. 3D) and extended erosions (19.6 %), and ring embedding (13.1 %) (Fig. 2C). located dorsally and ventrally to the radius and ulna, and also in the The recovered M. daubentonii with skin lesions showed an extended area most proximal area where the ring could be displaced. One forearm ring of skin thinning and depigmentation, and an erythematous plaque in the was found displaced from the propatagium, completely closed around distal carp (Fig. 2D). the radius and ulna, and embedded in the skin (Fig. 3E). After the Additional information on sex and reproductive state (for the latter removal, granulomatous tissue and inflammation were evidenced, and see Fig. A2, Appendix A) was available only for a subset of 304 bats the subcutis and muscles from the radius and ulna were exposed marked with forearm rings: M. schreibersii (n = 163), N. leisleri (n = 17), (Fig. 3F). Following the previous classification, 22 (n = 22, 100 %) bats and P. pygmaeus (n = 124). The results of the logistic regression model showed cutaneous lesions derived from the use of forearm rings, of using the previous subset of the dataset showed that the sex of the bats which six (27.3 %) had at least one mild lesion (Stage 1), 12 (54.5 %) did not have a significant effect on the probability of lesions. In contrast, presented at least one moderate lesion (Stage 2), and four (18.2 %) the reproductive state did, showing that bats in a passive reproductive showed at least one severe lesion (Stage 3). All cutaneous lesions state were less likely to develop skin lesions than those in an active (Table A2, Appendix A) were treated as needed and monitored until reproductive state (OR = 0.09, p-value = 0.018) (Table 2). The logistic their resolution. We examined all forearm rings (n = 22) after their regression model showed that P. pygmaeus had a significantly lower risk removal and recorded any signs of biting. Sixteen (72,7 %) forearm rings of developing skin lesions derived from the use of forearm rings than showed clear marks of bites (Fig. A3, Appendix A). M. schreibersii (OR = 0.38, p-value = 0.003) (Table 2). All tags were read correctly by the microchip scanner and found by palpation and skin transparency between the shoulder blades (Fig. A4, Appendix A) at the end of the study. None of the PIT-tagged bats (n = 21, 3.2. Impact of tagging C. perspicillata in captivity 0 %) showed cutaneous lesions by the end of the study. Bats marked with forearm rings presented a significantly higher frequency of lesions than 3.2.1. Skin lesions PIT-tagged bats (p-value < 0.0001). All banded bats (n = 22, 100 %) developed skin lesions of varying degrees of severity. Forearm rings were found displaced, piercing the dorsal and ventral aspects of the distal forearm in most banded 4 L. Lobato-Bailón et al. Biological Conservation 286 (2023) 110268 Fig. 2. Skin lesions found in free-ranging bats recovered with forearm rings in Catalonia. A. Callus formation (red arrow) in the dorsal aspect of the forearm of a Nyctalus leisleri. B. Ulcerated callus found in the forearm of a Pipistrellus pygmaeus. C. Marks of ring bite and ring embedding in the forearm of a Pipistrellus pygmaeus. D. Extended area of skin thinning and depigmentation (encircled), and an erythematous plaque (white star), in the distal carp of a banded Myotis daubentonii. 3.2.2. Behavioural changes and levels of cortisol metabolites in guano Table 2 We observed some of the banded bats biting the ring (Supplementary Summary of the logistic regression model with presence of lesion as the response Video C1, Appendix C) and scratching it with their hindlimbs in an and, species and reproductive state as predictors. The coefficients of the model are presented in terms of odds ratio along with their 95 % confidence interval. attempt to remove it (Supplementary Video C2, Appendix C). In Miniopterus schreibersii and active reproductive state are the reference categories contrast, no behaviours indicative of tagging-related discomfort were for species and reproductive state, respectively. Statistically significant values observed in PIT-tagged bats. are indicated with *. A statistically significant difference between the levels of CG before Variable OR 95%CI p-Value and after the intervention (Table B1, Appendix B) was found for group 1 (forearm ring, p-value = 0.013). In the remaining groups, the differences Intercept 72.87 [14.31, 1342.91] 0.0000 * were not statistically significant (Fig. B1, Appendix B). Nyctalus leisleri 0.97 [0.15, 18.77] 0.9763 Pipistrellus pygmaeus 0.38 [0.20, 0.71] 0.0026 * Passive reproductive state 0.09 [0.00, 0.43] 0.0182 * 4. Discussion Decades of individually marking bats with forearm rings have helped 5 L. Lobato-Bailón et al. Biological Conservation 286 (2023) 110268 Fig. 3. Skin lesions found in captive Carollia perspicillata due to marking with forearm rings. Areas of neovascularisation are indicated with yellow dashed arrows, and their respective brachial veins marked with arrow heads. A Skin depigmentation and thinning, dorsally and ventrally to the radius and ulna (encircled). An area of erythema is indicated with a white star. B. Skin depigmentation and thinning, dorsally and ventrally to the radius and ulna (encircled). An erythematous plaque is indicated with a white star. C. Dorsal cutaneous erosions, dorsally and ventrally to the radius and ulna (encircled). D. Cutaneous ulcer ventrally to the radius and ulna with active bleeding (white star) manifested after the forearm ring removal. Additionally, two punctiform erosions (red arrows) were present on the edges of the propatagium. E. Forearm ring displaced from the propatagium, firmly closed, and embedded in the skin (encircled). F. The same individual than E, after the forearm ring removal and disinfection of the wound. The subcutis and muscles from the radius and ulna were exposed and the cutaneous tissue around the ulcer was swollen and erythematous (encircled). us to understand many aspects of their ecology and biology (Ellison, et al., 2022; Zambelli et al., 2009), and other studies using bat bands 2008; Hutterer et al., 2005). However, the proportion of recovered bats whose authors noticed negative associated consequences and decided to is usually <1 % (Steffens et al., 2007), except for studies that work in report them (Balmori and Quetglas, 2000; Dietz et al., 2006; O’Shea roosting sites with high-fidelity species (Gaisler and Chytil, 2002). et al., 2004; Sendor and Simon, 2003). Our study confirms the health Therefore, recapture data accuracy relies on capturing and marking a impact of forearm rings in free-ranging bats, showing that a high per­ relatively large proportion of the population (Ellison, 2008; Hutterer centage (55.1 %) of bats recaptured in Catalonia over the last decade et al., 2005). had injuries derived from this marking tool. Moreover, considering the Knowledge of the physical impact of forearm rings has been obtained overall low recapture rate in bat mark-recapture studies, we must be by banding reports (Baker et al., 2001; Ellison, 2008; Hutterer et al., cautious in extrapolating the results to the whole population of bats. For 2005; Steffens et al., 2007), systematic studies in the wild (Mellado instance, it is likely that some of the non-recaptured bats had suffered 6 L. Lobato-Bailón et al. Biological Conservation 286 (2023) 110268 severe injuries from marking tools and died. This assumption is rein­ proportions of lesions compared to previous studies involving forced by the high percentages of C. perspicillata from our study in P. pygmaeus (Dietz et al., 2006; Sendor and Simon, 2003), M. schreibersii captivity that presented moderate (54.5 %) and severe (18.2 %) lesions (Baker et al., 2001) and N. leisleri (Zambelli et al., 2009). Variable di­ derived from the use of forearm rings. mensions of injury perception may lead to different conclusions between Factors reported to affect the level of injury caused by forearm rings researchers, particularly if their studies omit to report injuries. However, are the type of metal used, the accuracy of banders when placing the ring from a welfare point of view, no skin lesion regardless of its severity and the size of the ring (Baker et al., 2001; Dietz et al., 2006; Zambelli should be neglected but recognised as a source of physical discomfort to et al., 2009). Zambelli et al. (2009) reported skin lesions caused by bats. forearm rings in N. leisleri. They concluded that an incorrect band size Carollia perspicillata is one of the most studied bat species in the was the most likely reason for the injuries and suggested the use of laboratory and other captive settings due to its adaptability to captivity. slightly larger band sizes in case of doubt or in the absence of species- Furthermore, it is an abundant species in its indigenous habitat, so it is specific recommendations. Although we used a larger band size for also widely used in ecological studies (Rasweiler IV et al., 2009). N. leisleri bats, we still detected a high injury rate (53.3 %), even larger Because these studies may need to identify the bats individually, several than the 11.2 % reported by Zambelli et al. (2009). Furthermore, certain externally attached tools have been employed, including forearm bands morphological factors, such as the shape and width of the propatagium, (Alviz and Pérez-torres, 2020; Mellado et al., 2022; Monteiro et al., have been targeted as potential defining features for sensibility to bands 2019; Sánchez et al., 2007), plastic splits (Knörnschild et al., 2013; Rex (Dietz et al., 2006). However, these factors have not been accurately et al., 2011), and collars (Mellado et al., 2022; Monteiro et al., 2019; validated for European bats using long-term studies under captivity Rodríguez-Posada and Santa-Sepúlveda, 2013). However, despite the conditions and conclusions were based on field observations. Notably, a large body of scientific studies where the individual marking of high injury rate was reported by Dietz et al. (2006) in banded Rhinolo­ C. perspicillata is witnessed, there is often a lack of information regarding phus ferrumequinum, R. euryale and R. mehelyi, regardless of the size of the design or manufacturer reference (Alviz and Pérez-torres, 2020; the band or the existence of well-established recommendations. The Sánchez et al., 2007) and size (Alviz and Pérez-torres, 2020; Monteiro authors of the study concluded that the higher incidence of injuries in et al., 2019; Rex et al., 2011); and their physical impact or absence of these three species compared with those of banded vespertilionids injuries is hardly ever reported. All these factors significantly hinder resulted from increased ring rubbing along the frontal margin of the making an optimal choice in marking tools for C. perspicillata studies. propatagium due to a wider propatagium. In our study, only ves­ Similarly to Dietz et al. (2006), our study with captive C. perspicillata pertilionid species were assessed, but still, we found a concerning pro­ suggests that a wider propatagium may interfere with a proper slide of portion of injured bats. Since some of them are classified as cave- the band along the forearm. Slightly splitting the plagiopatagium and roosting species (e.g., M. schreibersii) and others as crevice-dwelling passing the ring through it has been used as an alternative for bats with species (e.g., P. pygmaeus), a larger impact was expected to be found large propatagia, such as C. perspicillata (Monteiro et al., 2019). How­ in the latter (Palomo et al., 2007). However, when the proportions of ever, this marking method is highly harmful, as bats are essentially left skin lesions were compared among our bat species, we found three with an open wound, susceptible to bacterial colonisation. Recently, species, N. leisleri, P. pygmaeus and M. schreibersii, exhibiting signifi­ Mellado et al. (2022) reported a significant occurrence of skin lesions in cantly worse responses to bands than the rest. For some of the species C. perspicillata resulting from this method. The healing process (in­ evaluated the sample sizes were really small, so larger studies are flammatory, proliferative and remodeling phases) may be prolonged needed. The logistic regression model accounting for the effect of sex and easily complicated when splitting the plagiopatagium, as friction and reproductive state showed that P. pygmaeus, our smallest crevice- between the wound edges and the ring is expected to be maintained as dwelling species assessed, was 62 % less likely to develop skin lesions long as the ring is fitted (Schultz et al., 2011). due to forearm rings than M. schreibersii. This result contrasts with the In our study with C. perspicillata, we found a significant difference in lack of a statistically significant difference between the two species in the levels of CG between the adaptation and the marking period in group the study using the complete dataset. This contrast can be attributed to 1, which was marked with forearm rings. However, the higher levels of the different methodologies employed and/or differences between the CG after the marking period cannot be explained by the marking tool complete dataset and the subpopulation used in the logistic regression alone because bats from group 2 (also marked with forearm rings) did model, involving factors that influence the risk of developing lesions. A not show the same tendency as group 1. Because cortisol secretion is a comprehensive understanding of the factors and variables influencing dynamic process (Busch and Hayward, 2009) and is individually the development of skin lesions is essential for a nuanced interpretation modulated by intrinsic factors such as age, reproductive state, or social of results. Future studies should incorporate larger datasets with a status (Palme, 2019), many factors may have jeopardised correct data comprehensive range of variables. Unlike males, female N. leisleri exhibit interpretation of the pooled group samples. We collected all faecal long-distance migratory behaviour (Tomás Alcalde et al., 2013), a trait samples at the same hour (in the morning) and CG in each sample rep­ that was previously assumed to cause worse consequences than seden­ resented an average of the defaecating period (12 h). During this period, tary habits. However, our results showed that sex does not seem to have different stressful events, such as social disputes, may have randomly a significant effect on the probability of developing lesions. In contrast, happened. Moreover, the temporal stability of cortisol metabolites we found that bats in an active reproductive state were more likely to shows significant variations among species (Donini et al., 2022). For develop skin lesions than those in a passive state. All these findings instance, the fast intestinal transit of C. perspicillata (Laska, 1990), emphasize that forearm rings have a significant impact on the health of coupled with differences in time deposition of faeces or low stability of insectivorous bats, regardless of their ecology, size, or sex. Furthermore, cortisol metabolites after deposition, could explain the absence of cor­ our findings highlight that during the reproductive season, bats may be relation in the remaining groups (Donini et al., 2022). Therefore, as a particularly vulnerable to banding activities. Skin lesion development first step, we recommend investigating the species-specific stability of due to forearm rings may be influenced by specific reproductive cortisol metabolites in faeces before any physiological study is per­ behaviour, hormonal fluctuations, immune system changes, or energy formed. Additionally, increasing the sample size and testing cortisol allocation during the reproductive season (Ruoss et al., 2019). Bats have metabolites levels individually -if possible- may be helpful to ameliorate a low annual reproductive output, compensated by a significantly longer the problem of high data variance in longitudinal studies where cortisol life span than other mammal species (O’Shea et al., 2004). Accordingly, changes in relation to an event are evaluated. any reduction in their reproductive success resulting from the conse­ Remarkably, there is no upper limit on the acceptable amount of quences of being banded may risk the bat population’s survival (Davy physical damage and pain that bats may experience due to marking et al., 2022; Schorcht et al., 2009). Overall, we found much larger tools. EUROBATS recognises that banding bats is an invasive procedure 7 L. Lobato-Bailón et al. Biological Conservation 286 (2023) 110268 that can seriously harm or even kill the bat if performed incorrectly. principles and evaluated by competent authorities and recognised in­ While recommendations of ring sizes by species are given, the Agree­ stitutions. We recommend that policymakers restrict the authorisation ment also states that sensitivity to bands may vary between species and of the use of forearm rings in bat species until adequate studies to also between individuals of the same species or population (UNEP/ evaluate their impact have been performed under controlled conditions. EUROBATS, 2018). Nonetheless, the decision of banding or not banding In any case, studies carrying out marking activities on bats should be bats rests on the researcher, as the establishment of a tolerable damage submitted to a legally constituted ethics committee for consideration, limit does. If we would follow the recommendation of Baker et al. (2001) guidance, and approval. to discontinue banding bats if injury rates surpass 2 %, banding activities would need to be terminated in most, if not all, insectivorous bat species. CRediT authorship contribution statement However, clinging to the principles of ethical research with animal subjects (Ethical Guidelines for the Use of Animals in Research, 2018), bat Lourdes Lobato-Bailón: Conceptualization, Methodology, Investi­ banding activities continue regardless of the evidence of physical in­ gation, Formal analysis, Supervision, Data curation, Writing – original juries because of the expected benefit obtained by this technique draft, Writing – review & editing. Adrià López-Baucells: Conceptuali­ (UNEP/EUROBATS, 2018). However, assumptions from these activities zation, Methodology, Investigation, Supervision, Data curation, Writing such as that marks are not lost with time or do not interfere with survival – review & editing. David Guixé: Conceptualization, Methodology, may introduce significant biases in bat population studies. Furthermore, Investigation, Data curation, Writing – review & editing. Carles Fla­ it is imperative that researchers minimise the risk of disturbance and quer: Investigation, Data curation, Writing – review & editing. Jordi suffering of bats and be responsible for considering options that may Camprodon: Investigation, Data curation, Writing – review & editing. improve the animal’s welfare while still obtaining the same benefit from Xavier Florensa-Rius: Investigation, Data curation, Writing – review & the research (Ethical Guidelines for the Use of Animals in Research, 2018; editing. Maria Mas: Investigation, Data curation, Writing – review & UNEP/EUROBATS, 2018). In our results and the reviewed literature, the editing. Laura Torrent: Investigation, Data curation, Writing – review & band injury rate for several species exceeded 10 % (Baker et al., 2001; editing. Laura Ordeix: Investigation, Writing – review & editing. Oriol Dietz et al., 2006; Mellado et al., 2022; Zambelli et al., 2009), which Tallo-Parra: Supervision, Writing – review & editing. Maria P. Ribas: points to considering other ethically acceptable alternatives. Investigation, Writing – review & editing. Ignasi Marco: Conceptuali­ In contrast, recent studies using PIT tags in free-ranging bats have zation, Methodology, Supervision, Writing – review & editing. Annaïs proved to be suitable for individual recognition (Locatelli et al., 2019; Carvajal: Formal analysis, Writing – review & editing. Manel López- van Harten et al., 2020), assessing bat movements (van Harten et al., Bejar: Formal analysis, Supervision, Writing – review & editing. 2019; van Harten et al., 2022a), reproductive success (Rigby et al., Sebastian Napp: Formal analysis, Writing – review & editing. Lola 2012), body condition (Locatelli et al., 2019; Rigby et al., 2012), and Pailler-García: Formal analysis, Writing – review & editing. Johan survival estimates (Ellison et al., 2007; van Harten et al., 2022b). This Espunyes: Investigation, Writing – review & editing. Oscar Cabezón: method has several advantages in comparison with the use of other Conceptualization, Methodology, Investigation, Supervision, Writing – traditional and long-term marking techniques. For instance, it provides review & editing. higher encounter probabilities with no handling or physical disturbance for species roosting in caves or bat boxes (Godinho et al., 2015; van Harten et al., 2019; van Harten et al., 2022b). Furthermore, there is no Declaration of competing interest evidence linked to detrimental effects on bats’ health, reproductive or survival success (Edmonds et al., 2017; Locatelli et al., 2019; Rigby The authors declare that they have no competing interests. et al., 2012; van Harten et al., 2020). This is supported by the results obtained in the present study. The use of a sterilised small-sized PIT tag Data availability injected subcutaneously in the dorsum caused no physical impact in any of our captive C. perspicillata bats. Four free-ranging vespertilionid No data was used for the research described in the article. species from Catalonia, including P. pygmaeus, whose body size does not exceed 4 g, neither showed lesions associated with this marking tool. Acknowledgements The present study highlights the potential of this technique to be used in bat species regardless of their morphological differences or body size. This work has received financial support from the Agència de Gestió Nevertheless, appropriate theoretical and practical training is crucial d’Ajuts Universitaris i de Recerca (AGAUR; 2020PANDE00028) and the before implementing any marking method. Fundació Zoo de Barcelona (ZOO2020_02). Maria P. Ribas was funded through the 2021 FI Scholarship, Departament de Recerca i Universitats, 5. Conclusions Generalitat de Catalunya, Spain (FI_B 00171). We thank all the veteri­ nary students and the Butterfly Park Empuriabrava staff for their Due to the high injury rates found in free-ranging and captive bats, collaboration and devoted care of the animals. banding activities are at odds with conservation goals and call into question the scientific value of recapture data obtained with this Appendix A. Supplementary data method. Our study concludes that the use of forearm rings should be banned under the ‘precautionary principle’ for all bat species until Supplementary data to this article can be found online at https://doi. systematic studies under controlled conditions are performed. Particu­ org/10.1016/j.biocon.2023.110268. larly, efforts should be made to include indicators of indirect effects such as changes in behaviour or reduced fitness. 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