Comparing the bacterial communities of wild and captive golden mantella frogs : implications for amphibian conservation Passos, LF, Garcia, G and Young, RJ http://dx.doi.org/10.1371/journal.pone.0205652 Title Comparing the bacterial communities of wild and captive golden mantella frogs : implications for amphibian conservation Authors Passos, LF, Garcia, G and Young, RJ Type Article URL This version is available at: http://usir.salford.ac.uk/id/eprint/48640/ Published Date 2018 USIR is a digital collection of the research output of the University of Salford. Where copyright permits, full text material held in the repository is made freely available online and can be read, downloaded and copied for non-commercial private study or research purposes. Please check the manuscript for any further copyright restrictions. For more information, including our policy and submission procedure, please contact the Repository Team at: [email protected]. RESEARCH ARTICLE Comparing the bacterial communities of wild and captive golden mantella frogs: Implications for amphibian conservation 1,2 2 1 Luiza F. Passos , Gerardo Garcia , Robert J. YoungID * 1 School of Environment and Life Sciences, University of Salford Manchester, Salford, United Kingdom, 2 Chester Zoo, Cedar House, Upton by Chester, Chester, United Kingdom * [email protected] a1111111111 Abstract a1111111111 Bacterial communities are frequently found in symbiotic associations with most animal a1111111111 a1111111111 species. The characteristically moist amphibian skin provides a good environment for the a1111111111 growth of some species of bacteria; among these a few can act as a first line defense mech- anism against infections. Amphibians in the wild have relatively high exposure to bacteria through environmental transmission and through interactions with different conspecifics, whilst in captivity animals interact with fewer individuals, as well as experiencing a less com- OPEN ACCESS plex environment through which to obtain their bacterial community. Here we compared the skin microbiota of captive and wild Mantella aurantiaca to investigate whether the captive Citation: Passos LF, Garcia G, Young RJ (2018) Comparing the bacterial communities of wild and environment was affecting individuals' skin associated bacteria. This could have survivor- captive golden mantella frogs: Implications for ship implications if captive animals had a different skin microbial community in comparison amphibian conservation. PLoS ONE 13(10): to wild counterparts and they were to be used in a reintroduction program. The microbial e0205652. https://doi.org/10.1371/journal. community were characterized through 16S rRNA amplicon sequencing methodology. Anal- pone.0205652 yses showed that captive individuals had significantly lower diversity of bacterial species Editor: Brenda A Wilson, University of Illinois at and lower relative abundant microbiota when compared to wild populations; this could result Urbana-Champaign, UNITED STATES in captive frogs released back to the wild probably has greater susceptibility to infections Received: October 13, 2017 due to inadequate skin microbiota. Accepted: September 28, 2018 Published: October 31, 2018 Copyright: © 2018 Passos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Introduction reproduction in any medium, provided the original The global amphibian crisis has resulted in increased use of captive breeding as a conservation author and source are credited. tool [1]. Maintaining captive populations is important in terms of species conservation for Data Availability Statement: All relevant data are potential reintroduction into the wild [2]. However, there is evidence that the captive environ- within the paper and its Supporting Information ment can have negative impacts on different aspects of amphibians' ecology and behaviour, files. such as affecting their vocalizations [3], anti-predator responses [4] and skin microbiota [5], Funding: LFP was in receipt of a CAPES which could potentially affect the survival of released animals. postgraduate scholarship Proc. 6060-13-7. Bacterial communities are commonly found in symbiotic associations with most animal Competing interests: The authors have declared species [6,7]. Frequently, the bacterial community provides some sort of advantage to the host that no competing interests exist. such as protection against pathogens [8], and in return, receives nutrients and a suitable PLOS ONE | https://doi.org/10.1371/journal.pone.0205652 October 31, 2018 1 / 12 Bacterial communities of wild and captive golden mantella frogs microhabitat in which to live and reproduce [9]. The characteristically-moist amphibian skin surface provides a fertile environment for the growth of bacteria [7], some of which may be present throughout the life of the organism, and some of which that will vary according to environment drivers and life stage [10]. These symbiotic bacterial communities contribute to the innate immunity of the host amphibian via competitive interactions between species and the production of antimicrobial metabolites, which are able to control the growth of some potential pathogens [11]. Thus, they play an important role in protecting amphibians from infectious diseases, such as chytridiomycosis caused by the pathogenic Batrachochytrium den- drobatidis [11,12]. This pathogen has been found around the world as well as in different areas in Madagascar, the natural habitat of our study species [13]. The microbiota of amphibian skin is one of the defense mechanism this group has against infections [8,9,14, 15]. Therefore, the proper functioning of this symbiotic interaction between bacteria and amphibians is vital for captive individuals, which are due to be released back into the wild [14]. To understand whether captive bred frogs are fit for reintroduction, in terms of their skin microbiota, wild and captive frogs of the same species need to be compared. Antwis et al [14] observed changes in the richness and abundance of microbiota of captive Agalychnis callidryas when compared to their wild counterparts and, a similar result was also found in six species of Japanese amphibians [9] and for the Panamanian golden frog, Atelopus zeteki [15]. Kueneman et al. [5] has demonstrated the effect of captivity on the loss of skin-associated bac- teria on frogs and increased chances of infections. The focus of this study is the golden man- tella frog, a critically endangered and endemic species from Madagascar, which will have captive bred individuals reintroduced to boost wild populations' numbers in a near future [16]. It is necessary to understand how captivity might have affect individuals to evaluate if ani- mals are suitable candidates prior to release. Amphibians in the wild have relatively high exposure to bacteria through environmental transmission and through interactions with both conspecifics and other species [17]. Amphibi- ans in captivity interact with fewer individuals, as well as living in a less complex environment in which to obtain a rich and diverse bacterial community [14]. Husbandry guidelines for keeping amphibians include removing waste, cleaning substrate and using a bleach dilution on enclosures to avoid the risk of diseases, but this could lead to a more sterile environment [18]. Consequently, captive amphibians are likely to be exposed to a lower diversity of bacteria, and thus support a much simpler skin-associated bacterial community in comparison to their wild counterparts. This could potentially make them less resistant to diseases when being reintro- duced to the wild environment [14,5]. During this research, we analysed how the unique set of conditions created by captive hus- bandry may affect golden mantella frogs' (Mantella aurantiaca) skin microbial composition [1±3]. We predicted that captive bred frogs will have a different bacteria composition with a less rich skin microbiota than their wild counterparts. Methods Ethical approval All the research reported in this study was approved by the Chester Zoo's Ethics Committee, Ambatovy, University of Salford Science and Technology Ethics Panel (ST1617-82) and, it conforms to all regulations and laws in all relevant countries in relation to care of experimen- tal animal subjects. Furthermore, we can confirm, from our post-experimental monitoring, that no animals suffered any injuries, became ill or were negatively affected as a result of this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0205652 October 31, 2018 2 / 12 Bacterial communities of wild and captive golden mantella frogs Study subjects The model species for this study was the golden mantella frog (Mantella aurantiaca). This is a species classified as critically endangered by the IUCN [19] and is endemic to the Mora- manga district, in the Region of Alaotra-Mangoro, Madagascar. Its distribution is restricted to a fragment of forest surrounded by degraded land. A significant proportion of its popula- tion is located inside or near the area of the Ambatovy mine [20]. Gold mantella frogs are well known due to their aposematic orange-red colouration. Females are characteristically larger and heavier than males [20]. Following a conservation needs assessment, the Amphib- ian Ark prioritised M. aurantiaca as a species in need of ex situ assistance to safeguard its survival [21]. Study sites The data used for this study were obtained from captive (Chester Zoo, UK) and