Microbial diversity on the skin of frogs


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Microbial diversity on the skin of frogs

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Title:
Microbial diversity on the skin of frogs
Translated Title:
Diversidad microbiana en piel de ranas
Creator:
Clow, Ashley
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Language:
Text in English

Subjects

Subjects / Keywords:
Frogs ( lcsh )
Ranas ( lcsh )
Fungi--Parasites ( lcsh )
Hongos--Parásitos ( lcsh )
Costa Rica--Puntarenas--Monteverde Zone
Costa Rica--Puntarenas--Zona de Monteverde
EAP Fall 2016
EAP Otoño 2016
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Reports

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Abstract:
Frogs interact with their environment directly through their skin through activities that are necessary for survival, such as respiration and rehydration. This causes vulnerability to pathogens like B. dendrobatidis, the Amphibian Chytrid Fungus, that can be picked up in the environment and passed between individuals. I ask if other types of fungi are present on the skin of frogs and whether they are picked up in the environment or living on the skin. To answer these questions, I swabbed 19 individuals from five species of frogs and cultured the fungi that I collected. Nine different types of fungi or fungal spores developed within 48 hours of incubation. After analyzing the data, I suggest that the microbes were picked up as the frog moves about its environment and do not grow on the skin. ( ,, )
Abstract:
Las ranas interactúan con su entorno directamente a través de su piel en actividades necesarias para su supervivencia, como son la respiración y la rehidratación. Esto provoca que las ranas puedan ser muy vulnerables a patógenos, como por ejemplo B. dendrobatidis, el hongo quitridio, que puede ser obtenido en el medio ambiente y contagiado entre los individuos. Me pregunté si hay otros tipos de hongos presentes en la piel de las ranas y si son recogidos en el medio ambiente o viven en la piel. Para responder a estas preguntas, realicé con suavidad frotis estériles de la piel de 19 individuos de cinco especies de ranas, y cultivé independientemente cada frotis en agar para desarrollar los hongos. Nueve tipos diferentes de hongos o esporas de hongos se desarrollaron dentro de las primeras 48 horas de incubación. Después de analizar los datos, sugiero que los microbios fueron recogidos por las ranas al moverse sobre su entorno y que no crecen en su piel.
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Student Affiliation: University of California, Santa Cruz
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Born Digital

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Monteverde Institute
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Monteverde Institute
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M39-00593 ( USFLDC DOI )
m39.593 ( USFLDC Handle )

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Microbial Diversity on the Skin of Frogs Ashley Clow University of California, Santa Cruz EAP Tropical Biology and Conservation Program, Fall 2016 16 December 2016 ______________________________________________________ ________________________ ABSTRACT Frogs interact with their environment directly through their skin through activities that are necessary for survival, such as respiration and rehydration. This causes vulnerability to pathogens like B. dendrobatidis the Amphibian Chytrid Fungus, that can be picked up in the environment and passed between individuals. I ask if other types of fungi ar e present on the skin of frogs and whether they are picked up in the environment or living on the skin. To answer these questions, I s wabbed 19 individuals from five species of frogs and cultured the fungi that I collected. Nine different types of fungi or fungal spores developed within 48 hours of incubation. After analyzing the data, I suggest that the microbes were picked up as the fr og mov es about its environment and do not grow on the skin. ______________________________________________________ ________________________ Diversidad Microbiana en Piel de R anas RESUMEN Las ranas interactan con su entorno directamente a travs de su piel en actividades necesarias para su supervivencia, como son la respiracin y la rehidratacin. Esto provoca que las ranas puedan ser muy vulnerables a patgenos, como por ejemplo B. dendro batidis el hongo quitridio, que puede ser obtenido en el medio ambiente y contagiado entre los individuos. Me pregunt si hay otros tipos de hongos presentes en la piel de las ranas y si son recogidos en el medio ambiente o viven en la piel. Para responde r a estas preguntas, realic con suavidad frotis estriles de la piel de 19 individuos de cinco especies de ranas, y cultiv independientemente cada frotis en agar para desarrollar los hongos. Nueve tipos diferentes de hongos o esporas de hongos se desarro llaron dentro de las primeras 48 horas de incubacin. Despus de analizar los datos, sugiero que los microbios fueron recogidos por las ranas al moverse sobre su entorno y que no crecen en su piel. ______________________________________________________ ________________________ Anurans, especially frogs, are extremely diverse and physiologically unique animals. 141 species of frogs and toads in Oder Anura are found in Costa Rica. This can be attributed to its unusual geolo gical history, varying geography, and the many environmental protection measures it has implemented (Chacn & Johnston, 2013) habitat provides sanctuary to 50 of the 141 species found throughout the country (Mont everde Tours, n.d.) What makes these creatures unique is the way in which they use their skin for survival. Like most animals, their skin acts as a protective barrier between their internal organs and the outside world. However, the skin of anurans, par ticularly frogs also contributes to respiration and rehydration. Frog skin is permeable to water which means that, rather than drinking water,

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Microbial Diversity on Frogs Clow 2 they absorb it through their skin (Leenders, 2001) For further aid in water uptake, frogs have a specialized or gan called the seat p atch located in the pelvic and femoral region of the animal body. It is highly vascularized and does the majority of water absorption in comparison to th e frog skin. Thus, frogs drink by sitting on damp substrate and other sources of water (Savage, 2002). In addition, t his permeability allows oxygen to enter the body through their skin aiding in respiration especially when underwater. F rogs are essentially wearing their lungs on their skin (Leenders, 2001). This permeability, however, leaves anurans vulnerable to water loss and subs equent dehydration as well as suffocation It also exposes them to pathogens found in the environment (Leenders, 2001 ) For example, a pathogen known as Ba trachytrium dendrobatidis a f ungus in the C hytrid family, has been found to cause massive amphibian die offs all over the world. The fungus attaches and grows on the skin of amphibians causing the host to be unable to breathe, thermoregulate, or re hydrate leading to the death of the host within a few weeks (Kolbert, 2014) According to Pounds et al. (2006), B. dendrobatidis likely caused the disappearance of Bufo periglenes famous Golden T oad that was last seen in 1989 Few studies have been conducted on frog skin and its ability to pick up microbes other than B. dendrobatidis However, and its ability to carry C h ytrid fungus I assume that frogs pick up other p articles as they move about. With this in mind, I want to ask the following questions: Are other fungi or their spores present on the skin of frogs? And if so, a re these fungi picke or do they ? Does substrate play a role in the types of microbes found? MATERIALS AND METHODS Collection The collection of specimen s took place at night between 6:00 P.M. and 8:00 P.M., when most frog species are active. My study sites were the Sanctuario Ecologico, Monteverde Institute, the Estacin Biolgica and Bajo del Tigre. With the help of an instructo r I looked for frogs in leaf litter, on low vegetation with broad leave s such as Heliconia sp. on the trunks or branches of trees, an d near sources of water. Onc e I located frogs, I photographed them to help with identification. Next, I caught the specimen by quickly and gently grabbing them with my right hand. I then adjusted them in my hand so that I held their posterior end under two fingers which made their skin accessible, but would not allow them to escape as well as prevented their limbs from breaking. Finally, my partner swabbed the specimen using two sterile cotton swabs which I placed in plastic bags labeled with specimen numb er, location, species, and date. A total of nine hours were spent searching for frogs among study sites To avoid contamination from frog to frog during the collection process, I used a new pair of latex gloves per specimen. The used/ possibly contaminated gloves were turned inside out and disposed of in a trash can with a lid. Then I put on a new, uncontamin ated glove before capturing my next specimen. This process was repeated with each capture. Lab Work To culture my fungus samples, I made agar using potato dextrose which I poured in petri dishes and stored in the refrigerator until fungal swabs were obta ined Once samples were

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Microbial Diversity on Frogs Clow 3 collect ed I brought them to the lab and swabbed the two swabs per frog on one agar plate and placed them in an incubator set at 37 degrees Celsius per instructions on the agar container I left each plat e in the incubator for 48 ho urs The incubation period allow ed the fungus enough time to grow without letting the specimen s overlap in the agar dish which would have caused difficulty in isolating them for microscope slides Following the incubation period I counted the number of different types of fungi in each agar dish and made microscope slides for each different type of f ungus using crystal violet stain. Finally, I photographed each slide through the microscope, recorded a description of the fungus, and placed th e agar dishes in the refrigerator to stop the growth process. To avoid contamination or spread of potential pathogens, I followed the agar disposal procedure provided by the Mont everde Institute. First, I soak ed all my petri dishes in a solution of 20% ble ach (1 part household bleach and 4 parts water) for at least 30 minutes. This sterilize d them by killing any chytrid or other containing substances on the dishes. Second, I rinse d them with water and wash ed with dish soap for future use. I made sure to sto re them in the glass cabinet with other s labeled sed dishes. dispose d of the sterilized agar in a labeled plastic container which will be discharged by staff RESULTS During the nine hours of looking for frogs I tested five species of frogs from two genera including four species of Craugastor and one species of Lithobates with a total of 19 individuals In order of abundance, the species consisted of 11 C. ridens four C. bransfordii two L. forreri one C. cruentus and one C. fitzengeri (Figure 1). As shown in Figure 2, most of the individuals, 16, were caught at the Santuario Ecologico which made up four of the nine hours of collection Only one individual was captured in each other location including Bajo del Tigre the Mo nteverde Institute, and the pond near the Estacin Biolgica I collected in each of these study sites between one and a half to two hours. During collection, I noted the type of substrate in which each individual was found. The re was a correlation between frog species and the substrate in which they were encountered (Table 1 ). The majority of C. ridens were located on the leaves of Bromeliads sp. or Heliconia sp. while C. bransfordii were found in leaf litter on the ground, and L. forreri were only encountered near ponds. After allowing my fungi time to grow for 48 hours in agar dishes, I found nine different typ es of fungi or fungal spores on the skin of my frogs which I classified based on their shape with a letter from A I (Table 2 ) L isted in order from A I, fungus shapes included, tiny circles, tiny ovals, pill elongate, string elongate, blue blotches, small ovals, tiny trans parent circles, clear circle chain and snowflakes (Appendix 1) Table 2 indicates the number of individuals per species that contained each type of fungus, but only presence among the species will be noted. Fungi A, B, and G were found in all five species. Fungus D was only found in Craugastor sp. Fungi C and E were found in e very species except for C. cruentus. Fungus F was only found in C. fitzengeri Fungus H was only found in C. cruentus. Fungus I was only discovered in one individual of C. ridens Overall, I noted that o ver 50% of fungus types were found on each species of frog Table 3 shows a comparison between each type of fungus found on the frogs and the types of substra te in which the frogs were caught Fungus A and B were found on every type of

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Microbial Diversity on Frogs Clow 4 substrate. Fungus C and E were found on frogs from every substrate except for the railing and the tree trunk. Fungus D was found on every substrate except for the pond. Fungus F and I were only found on leaves, while Fungus H was only found on the tree trunk. Fungus G was encountered on individuals from leaves, the tree trunk, and the pond. I noted that every fungus except for F, H, and I were found on over 50 percent of the substrates. Figure 1. Frog Species Tested : 19 total individuals from C. ridens, C. bransfordii, L. forreri, C. cruentus, and C. fitzengeri captured and swabbed 11 4 2 1 1 0 2 4 6 8 10 12 C. ridens C. bransfordii L. forreri C. cruentus C. fitzengeri Number of Individuals Species Frog Species Tested

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Microbial Diversity on Frogs Clow 5 Figure 2 Individuals per location : 19 individuals were captured and swabbed in four location s with the majority (16) from Sanctuario Ecologico. Table 1 Species vs. Substrate : Table indicates the type of substrate that each individual from each species was encountered. 0 2 4 6 8 10 12 14 16 18 Santuario Ecologico Bajo del Tigre Monteverde Institute Estacion Biologica/ Pond Number of Individuals Location Individuals per Location Species/Substrate C. ridens C. bransfordii L. forreri C. cruentus C. fitzengeri Leaf 9 1 Leaf litter/ ground 1 4 Railing 1 Tree trunk 1 Pond 2

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Microbial Diversity on Frogs Clow 6 Table 2 Fungus Type and Species : Table shows which fungus was identified on an individual from each frog species. The bold numbers in parentheses represent the total number of frog individuals swabbed within each species. Fungus type /Species A Tiny circles B Tiny ovals C Pill elongate D String elongate E Blue blotches F s mall ovals G Tiny transparent circles H Clear circle chain I Snowflake C. ridens (11) 9 4 8 4 3 3 1 C. bransfordii (4) 3 2 2 1 2 1 L. forreri (2) 1 1 2 2 1 C. cruentus (1) 1 1 1 1 1 C. fitzengeri (1) 1 1 1 1 1 1 1 Table 3. Fungus vs. Substrate : Table compares the fungus type found on each frog species and the substrate in which the frogs were found. Substrate/Fungus A B C D E F G H I Leaf X X X X X X X X Ground X X X X X Railing X X X Tree Trunk X X X X X Pond X X X X X DISCUSSION During nine hours of searching for frogs, I collected data from 19 individuals of frogs from two genera including Craugastor sp. and Lithobates sp. My data indicates that Craugastor sp. especially C. ridens which made up 11 of the 19, are the most common species I found in Monteverde. Of the four locations in which I collected frogs, 16 of the individuals were caught at the Sanctuario Ecologico, while only one individual was captured in each other l ocation. Although I collected at the Sanctuario Ecologico for two more hours than the other study sites, my results suggest th at it has the most abundant population of frogs among the four study sites In addition, I noted a correlation between species and the substrate in which they were encountered. My data suggests that certain species prefer specific microhabitats when they awaken at night. C. ridens prefer low lying vegetation such as Heliconia leaves, C. bransfordii thrive on the ground in dense leaf litter, and L. forreri only live near a large source of still water. Once I completed the lab portion of my project, I had collected nine different types of fungus from my frogs. Fungi A, B, and G were encountered on the majority of individuals from every species I swabbed. This suggests that these fungi are commonly found on the skin of frogs

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Microbial Diversity on Frogs Clow 7 in Monteverde. Fungus C and E were also commonly found among individuals, except for C. cruentus Because these fungi were so common among other species, the absence of Fungi C and E on C. cruentus could be a result of its small sample size of only one individual. Fungus D, was only found on species in Craugastor sp. This suggests that the skin of frogs in this genus are more suitable f or the fungus than that of frogs in Lithobates sp. The remaining fungi, F, H, and I, were only found on one individual in three different species with no correlation. Because over 50 percent of the fungi or fungal spores were found on each frog species but not every individual When I compared the nine types of fungus and the substrate in which I caught my frogs, I noted a correlation between some of the fungi and associated substrate. Fungi A and B were found on every type of substrate which indicate s that these fungi are ubiquitous in the environment. Each other fungi type, except for F, H, and I which were found on only one frog individual each, were encounte red on over 50 percent of the substrate types. This data suggests that frogs are picking up particles from their substrate; however, the substrate does not determine which fungi is picked up. It also indicates that the samples collected are fungal spores r ather than In conclusion, I found that every frog individual carried fungus or fungal spores on their skin. A lthough I noted a fungus correlation with each species if I found it on at least one individual, not ever y individual from a given species carried the fungus. This makes it difficult to pinpoint a clear correlation between fungus and frog species. This means that frogs are likely picking up fungi and their spores as they move about in their environment rather than having fungi that use frog skin as a microhabitat In addition, I discovered that substrate does not determine which microbes are found on the skin of frogs. This suggests that the fungi are in the form of fungal spores that are ub iquitous because th ey are spread throughout the environment through the air before settling on the substrate from which frogs pick them up. Very little is known about the particles that frogs are picking up on their skin as they move throughout their habitat. It is important to continue studying this topic because it could help to further understand the vulnerability of frogs and how they contract pathogens like B. dendrobatidis More research could possibly help with understanding how to combat such pathogens and perhaps prevent future outbreaks that could cause the disappearance of entire ACKNOWLEDGEMENTS Most importantly, I would like to thank Federico Chinch illa for mentoring me througho ut the course of my project, assisting me during several occasions to find and swab frogs and always reminding me to remain tranquila Huge thank you to Felix Salazar for inviting me to his home teaching me how to find frogs and assisting me with capturing them. Swibbity Swabbity, also like to thank the staff at the Monteverde Institute for allowing me to use the lab. Much gratitude to Sarah Callahan and Ronnie Appleseed for helping me learn how to make agar and slides and not getting too annoyed with my inexperience L ast but not least, thank you to my frogs for letting me swab you without too much difficulty

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Microbial Diversity on Frogs Clow 8 LITERATURE CITED Ch acn, F. M., & Johnston, R. D. 2013 Amphibians and r eptiles of Costa Rica. New Yo rk: Cornell University Press Kolber t, E. 2014 The sixth e xtinction: An unnatural h istory. New York: Picador Leenders, T. 2001 A guide to amphibians and r eptiles of Costa Rica South America: Distribuidores Zona Tropical Monteverde Tours (n.d.) Disappearing frogs of Monteverde Retrieved from www.monteverdetours.com/disappearing frogs in monteverde.html Pounds, J. A., et al. 2006. Widespread amphibian extinctions from epidemic disease driven by global warming. Nature 439 161 167. doi: 10.1038/nature04246 Savage, J. M. 2002 The amphibians and reptiles of Costa Rica Chi cago: University of Chicago Press APPENDICES Appendix 1. Fungus Shapes : Images A I show the different classifications of fungus based on their shape. Fungus A Tiny Circles Fungus B Tiny Ovals Fungus C Pill Elongate

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Microbial Diversity on Frogs Clow 9 Fungus D String Elongate Fungus E Blue Blotches Fungus F Small Ovals Fungus G Tiny Transparent Circles Fungus H Possible Chytrid Fungus I Snowflake


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