Establishment of the ex situ supporting program for vanished frog species that reappeared in Costa Rica: taking as model Lithobates vibicarius from Juan Castro Blanco National Park 1,2 1 3 Gilbert Alvarado Barboza , Federico Bolaños Vives & Sara González Camacho 1Biology School, University of Costa Rica, Costa Rica 2Department of Pathology (LAPCOM), School of Veterinary Medicine (FMVZ). University of São Paulo, Brazil. 3Laboratory of Biological Assays (LEBI), University of Costa Rica, Costa Rica. Total Funding requested from Amphibian Ark: USD $5 000. Executive summary: Costa Rica has been one of the countries that have reported the most important amphibian population declines, and it has been taken as a reference in iconic species such as the Golden Toad (Incilius periglenes). Fortunately, extinct species have reappeared in the last years. However, most of the efforts carried out at the moment have been in situ. In the last five years, the University of Costa Rica (UCR) has established good relationships with a conservative local NGO; a significant effort has been developed in the conservation of Lithobates vibicarius. Such effort has as main goal to turn this species in a conservation symbol of water in the north part of our country. This project’s mission is to develop infrastructure and human resources logistics to settle a back up colony of L. vibicarius, establish a physical facilities and training staff to give support in case of an emergency when any reappeared species in Costa Rica are in danger. To carry out this project, it was suggested to install two containers with the highest biosafety and animal welfare standards, backed up by the corresponding UCR committees. It will start with two L. vibicarius laying eggs, and the main goal will be to achieve the correct development of these animals’ life cycles until adulthood; the objective is to elaborate a breeding, maintenance and reproduction protocols of it. In addition, we will focus on developing methods that help reduce the impact of potential pathogenic agents; once this species is established in a controlled and healthy captivity environment, we will proceed to propose a reintroduction program to the natural sites where this species had completely disappeared. INTRODUCTION Since the late eighties, Costa Rica has been part of one of the main debates about amphibian population declines. Costa Rica’s most iconic species, the endemic golden toad Incilius periglenes, suddenly disappeared at the Monteverde Cloud Forest, and it was officially declared extinct in 2004. Nowadays, this is one of the symbols of extinct vertebrates around the world. Unfortunately, together with this species, many other Costa Rican amphibians started to disappear in the early nineties, especially those that belonged to high lands above 1000 masl. Among them, all the highland Rugulosus Group species disappeared, just as the Harlequin frogs of genus Atelopus and Incilius holdridgei. In the early 2000s, some populations started to reappear; it took more than a decade to see them again. Among them, there is the case of the Craugator rugulosus, Craugastor taurus, Incilius holdridgei, Agalychnis lemur, Agalychnis annae, Atelopus various and Lithobates vibicarius. Up to now, in the last five years, many efforts have been carried out in order to generate in situ information about these specimens, their population status and their health profile in regards to the presence of Batrachochytrium dendrobatidis (Bd) and Ranavirus. However, we have been constrained by just carrying out on fieldwork due to the absence of an adequate space that reaches all biosafety and animal welfare policies that are required when having animals in captivity. Among these relict populations, L. vibicarius is the most representative one; it is supported by a group of local people, who are part of a Pro-Development Association from the Juan Castro Blanco National Park (APANAJUCA, by its Spanish initials). They are making a significant effort to preserve the sites where some of these populations inhabit currently. Nonetheless, in the last three years of monitoring this population, we noticed that one of the lakes where these species reproduce was invaded by a human settlement; this lake is nowadays the habitat various domestic animals. The other lakes that are located around the National Park are taking the same risks because they are found in sites dedicated to agriculture and cattle industry. For these reasons, it cannot be dispelled the possibility of losing these breeding sites at any moment. During the seventies, one of the last highest wetlands of our country was drained for livestock purposes, and it was perhaps the main breeding area for the Vibicaria. We found out that two more lakes were drained in the last ten years; these sites are used nowadays for agriculture. Due to all these reasons, together with the presence of pathogenic agents in these areas such as Bd, this population faces a series of threats that may terminate their existence. It is risky to think that the few existing breeding places in the JCBNP may be sufficient to sustain this amphibian population; not to mention, the noticeable changes in our rainy season and temperatures that are indispensable during the breeding cycle of this species. L. vibicarius represents a unique opportunity to understand why this species, which was thought to be extinct, has survived and recovered in this site after its drastic population declines in Costa Rica during the 90´s. Along the last ten years, a priority list has been established for the species that require an ex situ husbandry as part of an integral conservation program to ensure their survival; this is through a significant effort made by the main stakeholders of our country. One of these species is the L. vibicarius; at an in situ level, a conservation program with a formal and legal agreement with a cooperating framework has been developed between APANAJUCA and the University of Costa Rica. Therefore, we would like to take advantage of this propelling and the opportunity this species offers to start together an amphibian conservation program ex situ for the L. vibicarius. This can also serve as model for others species that have reappeared. Our mission is to develop infrastructure and human resources logistics to settle a backup colony of L. vibicarius; in addition, set up a physical environment to give support in case of an emergency when relict population of species that reappeared in Costa Rica are in danger. We will also focus on the development of methods that may help reducing the presence of pathogenic agents; once this species is established in a controlled and healthy environment, we will propose a reintroduction program to the natural sites where this species had completely disappeared. The objectives of establishing an ex situ program for the L. vibicarius are as follows: Create physical facilities that reach all the biosafety and animal welfare requirements for settling the breeding colony of L. vibicarius in captivity. Generate skills in human resource for the maintenance of a breeding colony of L. vibicarius in captivity. Develop a breeding, maintenance and reproduction protocols of L.s vibicarius in captivity. METHODOLOGY Since 2013, we have been working on a framework agreement between APANAJUCA and the UCR. Such agreement was signed by both parties in April 2016. This will allow team work for both institutions in a legal way. APANAJUCA will support in obtaining two containers for the physical facilities set up of the ex situ program for the L. vibicarius in Costa Rica. Such containers will be placed at the Rodrigo Facio Campus in the University of Costa Rica, San Pedro – Montes de Oca. Placing containers in this area is more convenient because it is easier for staff of the Laboratory of Biological Assays (LEBI, by its initials in Spanish) and university students to work over there; these students are part of a scholarship program or are getting paid to give the necessary support, ensuring sustainability in a middle or long term. It is important to say that the university will provide water, electricity and safety. In addition, Gilbert Alvarado (who was awarded a scholarship from the UCR) will be working full time in this project related to this species; when he is finished with his PhD., he will work (as established in the agreement) at the Biology School in the University of Costa Rica in this long term project. Once containers are placed, modifications will be processed. One of the containers will be destined to work on frog investigations, and the other will be used for insect reproduction for feeding. The frog investigation container will be adapted properly; afterwards, electricity and plumbing will be installed, just as air conditioner and their corresponding water filters. Then, shelves, UV lights, normal lights, interchangeable hydraulic pipes and tanks for the animals will be installed. The total set up of both containers will be carried out accordingly to what it was suggested and recommended by Poole & Grow (2012) in Amphibian Husbandry Guide. The money requested from Amphibian Ark will be investing mainly in frog container. Biosafety must follow the regulations that any other laboratory from the University of Costa Rica accomplishes. This case will be specifically monitored by the biosafety committee of the LEBI, and it will be adjusted to what Poole & Grow (2012) has commanded. The container will have two different sections, one is a small room or cleaning room to get prepared for entering the laboratory and avoid the entrance of any contaminant agent to where animals are. Animal waste will be handled in this same lab, following the policies by the Institutional Committee of Solid Waste Handling from the University of Costa Rica in each of its sections (http://www.vra.ucr.ac.cr/node/205). Animal handling and use will be carefully monitored by the Committee on Care and Use of Animals (CICUA, by its Spanish initials), which already gave the corresponding permission.