Helmuth Malone 1 Microbial d iversity found on Anura in the Monteverde a rea Lilly Helmuth Malone Department of Ecology, Evolution, and Marine Biology Department of Molecular, Cellular, and Developmental Biology University of California, Santa Barbara EAP Tropical Biology and Conservation Program, Spring 2018 8 June 2018 ABSTRACT The microscopic world of fungi and bacteria have a significant effect on the organisms they grow on and can have either a beneficial or detrimental effect on said organism. Frogs are especially vulnerable to microbes due to their permeable skin, which leaves them susceptible to such pathogens as chytrid fungus. This project aims to discover the different types of fungal and bacterial morphologies found growing on frogs . In order to learn more about these microbes, I swa bbed 34 frogs from nine specie s f ound at four different locations in the Monteverde area and cultured the swab s on two different types of agar : potato dextrose agar and tryptone soy agar. I found 84 different fungal morphologies and 6 1 different bacterial morphologies aft er 48 hours of incubation at 30 C. After analyzing the data , I suggest that there is no clear correlation between morph great variation amongst the microbes g rowing on different species of frogs, and even amongst individuals of the same species of frogs. Diversidad m icrobiana e ncontrada en piel de a nur os en el Ã¡ rea de Monteverde RESUMEN El mundo microscÃ³pico de los hongos y las bacterias tiene un efecto significativo en los organismos en los que crecen, y puede tener un efecto beneficioso o perjudicial sobre dicho organismo. La s ranas son especialmente vulnerables a los microbios debido a su piel permeable , lo que las hace susceptibles a patÃ³genos como el hongo quitridio. El objetivo de mi proyecto fue descubrir los diferentes tipos morfologÃas de hongos y bacter ias que se encuentran en la piel de las ranas. Para aprender mÃ¡s sobre estos microbios, recolect Ã© isopados de 34 ranas de nueve especies , encontradas en cuatro ubicaciones diferentes en el Ã¡rea de Monteverde ; luego cultivÃ© los isopados en dos tipos diferentes de agar: agar de dextrosa de papa y agar triptofano de soy a. EncontrÃ© 84 morfologÃas de hongos diferentes y 61 morfologÃas de bacteri a s diferentes despuÃ©s de 48 horas de incubaciÃ³n a 30 C . DespuÃ©s de analizar los datos, sugiero que no existe una correlaciÃ³n clara entre las morfologÃas que crecen en la piel de una rana y la especie de rana ; ademÃ¡s, hay una gran variaciÃ³n entre los microbios que crecen en diferentes especies de ranas, e incluso entre individuos de la misma especie de ranas. The study of animal microbe inter actions is an area of active research that has serious implications for the survival of many species. Microbes have evolved symbiotically with multi celled organisms and are present in all areas of life; there are an estimated ten times as many microbial cells than human cells in the human body ( Eisthen and Theis 2016). Studying these
Microbial Diversity found on Anura Helmuth Malone 2 interactions is important not just for a better understanding of the animal microbe interaction but also for human benefit as we ll. Over 40% of compounds used by modern medicine are de rived from nature (Groot et al 2012), signifying the importance of not only preserving the natural world but also continuing to study it. The secretions found on the skin of frogs, as well the microbiota living in symbiosis there, may also have potential health benefits that are relevant to society. order , Anura . The ir skin is permeable to water and p rovides protection against abrasion and pathogens, serves as a respiratory membrane, absorbs and releases water, and may contain poisons that are deleterious t o predators (Stebbins & Cohen 1995 ). The mucous gland secretions help to keep the skin moist and may pro vide protection against the entry of bacteria and molds ; some of these secretions have even been found to have antibiotic properties (Stebbins & Cohen 1995 ). Indeed, a study done t hat collected the skin secretions from around 50 frogs in Colombia found that some of these compounds displayed very strong antibacterial activity without being toxic to somatic cell lines (Groot et al 2012). Other examples of these secretions include a peptide isolated from three closely related Australian tree frogs th at shows antibiotic and antiviral activity (Stone et al 1992) and t he secretion of the Phantasmal Poison Arrow Frog (now known as epibatidine) that has been found to be more effective than morphine at blocking pain ( Fitch et al 2010 ). The peptide secretio n of the large Bicolored Tree frog is the first substance that has been found to interact with the adenosine receptor system, which is responsible for many cell communications and has implications in many brain dis et al 1992 ). These are just a few of the compounds found on frog skin that have implications for humanity. A mphibians face a declining population worldwide , mainly due to habitat fragmentation , pollutants, climate change, and disease . One of these diseases is chytrid f ungus ( caused by Batrachochytri um dendrobatidi s , or Bd ), which has been decimating frog populations across the world , including in the Mont e verde area. It thrives in cool, moist habitats and inhabits the skin of amphibians , causing the thickening of the mucosal secretions. This can lead to death as it disrupts the fluid/gas exchange abilities of the skin. The disease can cause massive die offs of frog populations and first appeared in Costa R ica in the late 1980s (Leenders 2016 ). However, there are some frog populations that survive an outbreak of Bd , mostly due to the presence of beneficial microbes. A study done on the effectiveness of symbiotic bacteria on frog skin found that these bacteria can provide protection against Bd as long as they can persist in the presence of the mucosal peptides secre ted by the frog (Woodhams 2007) , proving that there is hope in the fight against this deadly disease. Recently, a study published in Science used whole genome sequencing to trace the origins of chytrid fungus to the Korean Peninsula, where an ancestral population was found that emerged in the early 20 th century. This coincides with the rise of the global trade of amphibians and thus helped to spread this devastating disease worldwide and contri bute to glob et al 2018). Further research is necessary in order to determine other types of microbes residing on frog skin and the effects these microbes may have on the frogs. There are 147 species of frogs and toads found in Costa Rica, and of those, 51 species are found in Monteverde (Hayes et al 1989 ) , making this an opportune location to study the m and learn more about the microbes living on them . Between the 1980s 2000s, over 20 s pecies of frogs disappeared from the a re a and s ince then, roughly half have repeated, with the search for the other species ongoing ( Leenders 2016) . While hunting for frogs is a very entertaining and
Microbial Diversity found on Anura Helmuth Malone 3 adventurous process, there is an entire world lying just on the surface of th e se multi colored amphibians. A study done by McKenzie et al in 2012 found that amphibian host species was a high predictor of bacterial morphology similarity between individuals and that innate frog species differences may regulate the colonies of microbes f ound on each frog . My goal is to determine how the microbio ta inhabiting frog skin varies between different species and also between individuals from the same species. MATERIALS AND METHODS Lab Work I made agar plates in order to provide a nutrient rich living situation for all the microbes I would find on frogs . I washed g lass Petri dishes with soap and water and then sterilized them in a pressure cooker . I chose to use Potato D extrose A gar (PTA) to grow any fungal microbes fou nd on the frog and Tryptone S oy A gar (TSA) to grow any bacteria found on the frog. To make PTA, I added 15.8 grams of agar to 400 mL of deionized water, which was then microwaved until bubbles were seen . The agar was left to cool for ~15 minutes and then poured into 12 glass Petri dishes. To make TSA, I added 16 grams of agar to 400 mL of deionized water and the above process was repeated. After letting the agar solutions cool, I labeled the plates with their respec tive type of agar and placed them upside down in the fridge until needed. I made m ore agar plates as necessary by repeating this process. Field Work The second step was f in ding frogs. I swa bb ed frogs from four different locations: Santuario Ecologico , Bajo del Tigre, Estaci Ã³n BiolÃ³ gica Monteverde, and Selvatura Adventure Park. A total of 17 hours was spent looking for frogs between the hours of 3:30 PM 10:00 PM ( Appendix 1) . Weather varied at each location, depending on the day. After a frog was located, I placed the frog in a Ziploc bag in order for ease of swabbing . I used g loves at all times to handle the frog, and a new pair of gloves was used for each frog. After the frog was placed in the bag, I swabbed it on the dorsal side using two sterile swab s. The frog was then released in the same spot and the swab s sealed in the Ziploc bag. Each frog was captive for less than five minutes and all 34 frogs were released alive. I took p ictures before or after swabbing , dep ending on weather conditions and feistiness of the frog. I used n ew Ziploc bags for each frog. Each bag was labeled with the date, time, location, and type of frog swabbed . After collecting the frog skin microbial samples , I took the Ziploc bags back to the laboratory at the Institute the same night in order to prevent future contamination of the swabs . The one exception to this was the last night of frog hunting ( 23 May 2018 ) as we finished late, and the lab closed at 10 PM, so these swab s were plated the next morning at 8 AM. I removed t he agar plates from the fridge and plated o ne swab from each frog on a PTA plate and the other swab on a TSA plate. I plated s wab s by slowing drawing the swab back and forth across the plate in a zig zag manner . I taped t he plates shut and placed them upside down in the incubator for 48 hours, set at 30 C . After two days, I removed the plates from the incubator and either looked at them the same day or placed them in the fridge in order to halt growth (this was done if there were too many samples, or if it was getting close to 10 PM). I looked at e ach plate under the dissecting microscope in order to classify the different types of fungal and bacterial colony morphologies
Microbial Diversity found on Anura Helmuth Malone 4 that were presen t on the agar plate . I recorded t he total number of morphologies, as well as morph type, f or each plate. I took ph otographs of each morphology in order to create a reference guide (see Appendix 2 and Appendix 3 for this guide) . After I observed the plates , I left them in a bucket of 20% bleach for 24 hours in order to sterilize any microbial growth. I then washed t he glass Petri dishes with soap and water in order to be reused and disposed of the agar i n the liquid waste bin. The gloves, Ziploc bags, and swabs were all disposed of in the Biohazard Waste bin. To coefficient: where a represents the number of species in both samples, b represents the number of species unique to one sample and c represents the number of species unique to the other species (Krebs, 1989). This value can vary from zero to one, with zero representing no similarity and one representing complete similarity be tween morphologies. For this study, I considered any value above 0.75 to be high similarity and any value below 0.25 to be low similarity. RESULTS A total of 34 individual frogs from nine different species were swabbed ( Figure 1 ). After allowing the mi crobes to grow on the agar, a total of 84 different fungal morphologies were found on the potato dextrose agar ( Appendix 2 ) , and a total of 61 different bacterial morphologies were found on the tryptone soy agar ( Appendix 3 ) . Due to a delay in the making of the TSA, there is no bacterial morphology data for the first three frogs swabbed (two P. ridens and one R. forreri ). There was an average of 9.5 fungal morphologies growing on each frog, based off the total number of morphologies discerned on the PTA and an average of 8.2 bacterial morphologies growing on each frog, based off the total number of morphologies discerned on the TSA. The frog with the most fungal morphologies, as well as the frog with the least fungal morphologies, were both found right after the rain. The same was true when comparing bacterial morphologies. The frogs found at the pond had a considerably lower average number of fungal morphologies per individual (5.72) compared to the overall average but had a higher average number of b acterial morphologies (9.0) compared to the overall average .
Microbial Diversity found on Anura Helmuth Malone 5 Figure 1: Occurrence of each frog species . A total of 34 individuals were swabbed . E ach fungal morphology appeared on an average of 3.8 individual frogs and each bacterial morphol ogy appeared on an average of 4.2 individual frogs. Total fungal morphologies found on each frog varied from one (frog #31) to 23 (frog #2). Total bacterial morphologies found on each frog varied from four (frog #28 and #34) to 13 (frog #5) . C. stejnegarianus had t he largest number of fungal and bacterial morphologies found in total, but C. fitzingeri had the highest average ( Figures 2 &3). Data for any frog species that was only swabbed once was discounted due to lack of sufficient data. Figure 2: Total types of fungal morphologies found on each frog species, along with average number of morphologies found per each individual frog for that species . 0 10 20 30 40 50 60 Fungal Morphologiies Found Species Total Morphs Average 0 2 4 6 8 10 12 14 Number Species
Microbial Diversity found on Anura Helmuth Malone 6 Figure 3 : Total types of bacterial morphologies found on each frog species, along with average numbe r of morphologies found per each individual frog for that species . No data was recorded for R. forreri as the TSA had not been made in time. A key was used to identify morphology us ing the physical appearance of each colony morph in order to describe and name the morph. F ungal morph six (dark grey circle) was the most common to observe , followed by morph two (yellow small circle) and morph 52 (small black spot). However, it was most common to see each fungal morphology only once ( Table 1 ). B acterial morph L (grey circle) was the most common to observe, followed by morph C (suspended triangle spot) and morph D (suspended circle dot). As with the fungal morphologies , it was most common to see each bacterial morphology only once ( Table 2 ). Th e only fungal morphology that occurred across all nine species was morph 6; no bacterial morphology occurred across all nine species, although morph AN and morph C occurred on seven of the nine species. 0 5 10 15 20 25 30 35 40 45 50 Bacterial Morphologies Found Species Total Morphs Average
Microbial Diversity found on Anura Helmuth Malone 7 Table 1 : Occurrence of each type of fungal morphology found across individual frogs. It was most common to see each morphology only once or twice. Morph 6 was the most common, occurring on 26/34 individual frogs. Refer to Appendix 2 for a thorough list of morphologies. Fungal Morphology O ccurrence 13, 15, 25, 29, 41, 46, 50, 54, 59, 60, 61, 62, 64, 67, 69, 74, 75, 76, 77, 80, 82, 83, 84 1 8, 10, 14, 16, 21, 24, 36, 37, 40, 47, 48, 55, 57, 66, 68, 70, 72, 78, 79, 81 2 7, 17, 26, 33, 49, 51, 58, 63, 71 3 4, 9, 22, 23, 27, 31, 34, 38, 43, 56, 65, 73 4 1, 32, 39 5 12, 45 6 3, 28, 35, 53 7 5, 11, 18, 19, 20 8 30, 44 9 42 10 52 15 2 21 6 26 Table 2: Occurrence of each type of bacterial morphology found across individual frogs . It was most common to see each morphology only once or twice. Morph L was the most common, occurring on 22/3 1 individual frogs. Refer to Appendix 3 for a more thorough list of morphologies. Bacterial Morphology Occurrence B, J, W, X, AA, AK, AO, AQ, AS, AV, AW, AY, BA, BD, BE, BG, BH, BI 1 O, P, T, AE, AG, AM, AR, AT, BC, BF 2 A, Y, Z, AC, AH, AI, AJ, AU, AX 3 H, AL, AP, AZ 4 R, V, AB, AF 5 G, I, N, U, AD, BB 6 K, S 7 E, F, Q 8 AN 9 M 15 D 16 C 17 L 22 In order to compare the morphologies found between coefficient of similarity to determine the similarity that occurred in both groups of frogs being discussed . I first compared the similarity in morphologies between members of I. p seu dopuma
Microbial Diversity found on Anura Helmuth Malone 8 found on leaves vs. found in the pond at the Station and determined there to be little similarity in fungal morphologies and only slightly higher similarity in bacterial morphologies (Table 3 ). I. pseudopuma found on lea ves versus found in the pond . The top value is based off fungal morphologies and the bottom value is based off bacterial morphologies. FUNGAL I. pseudopuma Leaf # of morphs present # of morphs absent Pond # of morphs present 2 14 # of morphs absent 3 X S s = 0.19047619 BACTERIAL Leaf # of morphs present # of morphs absent Pond # of morphs present 5 16 # of morphs absent 2 X S s = 0.357142857 I then did a comparison between members of P. r idens and I. pseu dopuma found on leaves, determining there to be higher a similarity in this comparison for both fungal morphologies and bacterial morphologies (Table 4 ) compared to the above similarity comparison. I. pseudopuma and P. ridens found on leaves. The top value is based off fungal morphologies and the bottom value is based off bacterial morphologie s. FUNGAL I. pseudopuma # of morphs present # of morphs absent P. ridens # of morphs present 4 4 # of morphs absent 29 X S s = 0.195121951 BACTERIAL I. pseudopuma # of morphs present # of morphs absent P. ridens # of morphs present 6 11 # of morphs absent 1 X S s = 0.5 My final similarity comparison was between the three species with the highest occurrences : C. stejnegerianus , I. pseudopuma, and P . ridens (Table 5 ) . I chose to only compare data between these three species in order to increase sample size and get a wider variety of morphologies. C. stejnegerianus and P . ridens had the highest fungal morphology similarity and P . ridens and I . pseudopuma had the highest bacterial morphology similarity . Further comparisons in similarity between intra genus species reveal that the two Craugastor species share more similarities in both fungal and bacteria morphologies than the two Pristimantis species ( Table 6 ).
Microbial Diversity found on Anura Helmuth Malone 9 between the three most common species. The top values are based off fungal morphologies and the bottom values are based off bacterial morphologies. FUNGAL C. stejnegerianus I. pseudopuma P. ridens C. stejnegerianus 1 X X I. pseudopuma 0.263 1 X P. ridens 0.553 0.321 1 BACTERIAL C. stejnegerianus I. pseudopuma P. ridens C. stejnegerianus 1 X X I. pseudopuma 0.418 1 X P. ridens 0.328 0.6 1 Craugastor and between two species of Pristimantis. The top values are based off fungal morphologies and the bottom values are based off bacterial morphologies. FUNGAL C. stejnegerianus P. cruentus C. fitzingeri 0.423 X P. ridens X 0.213 BACTERIAL C. stejnegerianus P. cruentus C. fitzingeri 0.531 X P. ridens X 0.370 DISCUSSION Morphology Comparison At the end of this project, I found 34 total frog individuals which had a combined 84 different fungal morphologies and 6 1 different bacterial morphologies. Looking at two previous studies done on frog microbes in the Monteverde area ( Chow 2016; Desales 2017), I found a larger population of microb ial in comparison . A study done that experimentally infected frogs with the chytrid fungus ( Bd ) found that during the warm wet season, frogs limited Bd infections and recruited potentially beneficial bacteria, resulting in a higher diversity of bacterial colonies, while during the cool dry season, Bd infections increased with time and bacterial diversity rem ained constant (Longo & Zamudio 2017). This co uld explain the difference between the during the Fall ( cool dry season) program . Spring (warm wet) program, which I wo uld suggest should result in similar results. While the two previous studies did use different incubation temperatures , which would have resulted in a different possible set of microbes, I would still expect to get a similar number of morphologies . As I had a larger sample size compared to these two studies, I would hypothesize that this was a contributing factor to the large number of morphologies I found.
Microbial Diversity found on Anura Helmuth Malone 10 The individual frog with the most fungal morphologies was a Pristimantis ridens , which is a type of rain frog commonly found on leaves. I expected that a species of Craugastor , or litter frog, would have the most morphologies as these frogs are commonly found in the leaf litter on the ground (Leenders 2016) . Leaf litter tends to have more microbes in it than other substrate places due to the additional processes that occur there such as decomposition and the recycling of nutrients which results in the habitation of more microbes compared to drier surfaces. However, the next five frogs with the most fungal morphologies were from Craugastor , which confirms the idea that these frogs, due to residing in the leaf litter, pick up more microbes on their skin. For bacterial morphologies, it was a Craugastor frog ( C. stejnegerianus ) that had the most indivi dual morphologies, which was what I expected. I thought that frogs found during the rain would have less microbe colony morphologies due to the wet weather washing some of the microbes away . H owever, after observing weather data I collected each night of frog hunting, I determined that the frogs found during the rain had a similar average compared to the overall average of number of fungal morphologies found per frog and even had a higher average compa red to the overall average of number of bacterial morphologies . So, rain could introduce additional bacterial colonies onto the frog skin that were not present in the environment before. Another interesting observation was the fact that the frogs found a t the pond had a lower average number of fungal colony morphologies compared to the overall average, but a higher average number of bacterial colony morphologies compared to the overall average. One paper describes how bacteria can inhibit the pathogenic effects of fungi f ound in the environment (Wargo & Hogan 2006), suggesting that there could be more fungal microbes found in the pond environment that necessitate a higher diversity in bacterial colonies found on the frog skin. While I did not compare m icrobe, morphologies found on the frogs in relation to the location they were found (at either Bajo del Tigre, Santuario , EstaciÃ³n , or Selvatura), I would , especially in terms of microbes the frog could pick up from the environment. This could be a potential area of future research in order to do learn more about the microbes found on frogs compared to the environment they inhabit. As many of the fungal an d bacterial morphologies occurred only once, it is hard to find any clear correlation between a specific microbe and the frog species it grows on. While there were a few morphologies that occurred across all, or most, of the frog species , I conclude that each individual frog had variable morphologies compared to other individuals. A study done that compared cohabiting bullfrogs and newts in the same pond determined that each species had its own distinc t microbial communities (Walke et al 2014) . I did find similar results, as there was little similarity between each species of frog, although there was also great variability in microbial morphologies in individuals from the same species . That same study also found that there was little overlap bet ween common microbes found on the amphibians compared to common microbes found in the environment, dictating that the amphibian skin may select for microbes that are not as common in the environment, as well as increase the variability of the microbes foun d on the skin (Walk e et al 2014). as higher variability in beneficial microbes found on a frog leads to a higher chance that the certain individual can survive against pathogenic microbes. Another study found that frogs that had higher bacterial diversity had lower Bd infections (Longo et al 2015), further proving the importance of having high variability and diversity in the microbiota that reside on frog skin.
Microbial Diversity found on Anura Helmuth Malone 11 Additionally, this is evidence of the eff ect that fungal bacterial interactions can have on a frog; in this case, these interactions prove beneficial. One fact to note is that, while PTA was chosen in order to specifically grow fungi and TSA was chosen in order to specifically grow bacteria, ther e is a possibility that the opposite microbe grew on the agar. For example, both PTA and TSA had a morphology known as morph 66, TSA morph BE) that looked very similar. I considered this fact, but without more detailed analysis of e ach morphology found and comparison using more intensive microscopic techniques, it is not possible to determine if there was any crossover between the two different agars. Therefore, I kept the morphology lists separate based on what type of colony was g rowing on each respective agar . Additionally, while I was certain to be careful while handling all the materials and tried to keep all my supplies in a sterile environment, there was the possibility of cross contamination by external microbes from the environment. However, this study did not include a control plate that would have accounted for this difference. Similarity Analysis Most comparisons between the three most common frog species swabbed resulted in similarity values between 0.2 0.4, which I interpreted to mean low to moderate similarity. However, the comparison of fungal morphologies between C. stejnegerianus and P. ridens had a similarity value slightly above 0.5, which I interpreted to mean slightly higher moderate similarity. Additionally, the comparison of bacterial morphologies between P. ridens and I. pseudopuma had a similarity value of 0.6, which I interpreted to mean moderate to high similarity. As both P. ridens and I. pseudopuma are mostly leaf dwelling species and are commonly fo und on low vegetation (Leenders 2016), I would expect these two species to have a higher similarity coefficient compared to C. stejnegarianus , which was true for bacterial morphologies. The fact that C. stejnegarianus and P. ridens had a higher similarity value for fungal morphologies could be because P. ridens can also be found in leaf litter during the day, while I. pseudopuma is almost strictly ve getative and arboreal (Leenders 2016). However, these differences in morphologies could al so be due to random chance. I did two intr a genus comparisons to determine if species in the same genus had high or low similarity in microbial morphologies present . The two species of Craugastor compared had similarity values close to 0.5 for both bacterial and fungal morphologies, representing a moderate level of similarity. This was not the case with the two species of Prist i mantis compared which had similarity values between 0.2 0.4 , representing a low to moderate level of similarity. Based off this data, it is not possible to state that individuals of the same genus share more similar morphologies compared to individuals from different genera due to the fact that neither comparison showed high levels of similarity. This s upports my conclusion that there is great variability amongst the microbes found on frogs, no matter the close relation between them . My last similarity analysis was to determine the effect substrate has on microbial morphologies found on a frog . While three of these comparisons resulted in values less than 0.5, the comparison of bacterial morphologies between individuals of I. pseudopuma and P. ridens found on leaves did have a significant value of 0.5, suggesting moderate similarity between these two populations. This indicates that substate does play a role in the types of morphologies present ; in this case, substrate was a better indicator of similarity in colony morphology than species was, which is the opposite result compared to the 2012 stu dy that found that frog species was a high indicator of bacterial colony similarly between individuals (McKenzie et al 2012).
Microbial Diversity found on Anura Helmuth Malone 12 Another study found that on substate location and could be a possible reason for the differences in similarity o bserved in my study (Kueneman et al 2014). Interesting Results One interesting result I found is from f rogs 18 and 19 ( C. fitzingeri) which were found during amplexus. I would expect these two frogs to have similar microbial morphologies present due to the close contact of the frogs. However, only three fungal morphologies and five bacterial morphologies were shared between the two frogs. This may be because I swabbed only the backs of the frogs; if I had instead swabbed the stomach of the male frog, it may have had more similar morphologies compared to the female frog due to the close contact of the female back and male stomach achieved during amplexus. An other interesting result that I found occurred on sample 2 (PTA) , sample 5 (TSA), and sample 24 (TSA). Both had plenty of microbial growth; however, there were inhibition circles around some of the microbes that disrupted this growth . An inhibition circle results when there is some sort of compound or chemical that prevents a certain microbe from growing. This is especially interesting as it means there are microbes either growing on the frog or picked up by the frog from its environme nt that inhibit other microbes found on the frog. While the individual bacterial interactions For example, the bacterial species Janthinobacterium lividum , which is found on the skin of several species of frogs, can prevent death caused by Bd when added to the skin of Rana mucosa (Harris et al 2009). These microbes can be a determinate of disease outcome and thus offer another reason to learn more about the microbiota inhabiting frog skin. Further Research Further research is needed to learn more about the various microbes found on the frogs, and any possible effects these microbes could have on the fro g. This is an important area of research as not much is known about the se different microbes (fungi or bacteria) that are found or picked up by frogs from the environment . Some of these microbes, such as Bd , may have a deleterious effect on the frog, but o thers may have beneficial effects that instill protection to the frog, allowing the frog and microbe to live in harmony with each other . The variation I found in microbial colony morphologies indi cates that there is a diverse population of microbes that i nhabit frog skin and may contribute to the survival of the frog. Some of the microbes and medical field and human health, representing how important it is that researchers continue to learn more about the microbes found on frogs and ho w they can be used. ACKNOWLEDGEMENTS I would like to thank Federico Chinc h illa and Emilia Triana for advising me throughout the course of this project and for assisting in the finding and swabbing of the frogs. Thank you to everyone who helped in the search for frogs and with their identification: Christopher Salazar, Felix Salazar, and Mark Wainwright . Thank you to Luisa Moren o and the entire staff at the Monteverde Institute for a llowing me to use the lab, especially during those late night hours.
Microbial Diversity found on Anura Helmuth Malone 13 Than you also to the staff at Bajo del Tigre, Santuario Ecol Ã³ gico , Monteverde Biology Station, and Selvatura Adventure Park for allowing me to frog hunt on their properties. Thank you to my fellow EAP students who worked in the lab for providing entertainment during the long and rainy lab days. Thank you to Alex Lee for reviewing my paper as well as to Fede for helping in the translation of my abstract into Spanish. Most i mportantly, thank you to all the frogs I swa bbed for allow ing themselves to be captured and photographed and yet still managed to look so cute. LITERATURE CITED Chow, A. (2016). Microbial Diversity on the Skin of Frogs. UCEAP 2017. Daly, J., Caceres, J., Moni, R., Gusovsky, F., Moos Jr., M., Seamon, K., Milton, K., and Myers, C. 1992 . Frog secretions and hunting magic in the upper Amazon: identification of a peptide that interacts with an adenosine receptor. PNAS, 89 (22), 10960 10963. Doi: 10.1073/pnas.89.22.10906 . Desales, M.A. 2017. Frog skin harbors an abundance of fungal spores. UCEAP 2017. Eisthen, H. L., and Theis, K. R. 2016. Animal microbe interactions and the evolution of nervous systems. P hil. Trans. R. Soc. B. 371: 20150052. Doi: 10.1098/rstb.2015.0052 . Fitch, R., Spande, T., Martin Garraffo, H., Yeh, H., and Daly, J. 2010. Phantasmidine: An Epibatidine Congeners from the Ecuadorian Poison Frog Epipedobates anthonyi. Journ al of Natural Products, 73(3), 331 337 . Groot, H., Munoz Camargo, C., Moscoso, J., Riveros, G., Salazar, V., Kaston Florez, F., and Mitrani, E. 2012. Skin micro organs from several frog species secrete a repertoire of powerful antimicrobials in culture . Journal of Antibiotics, 65, 461 467. Harris, R.N.2009 , Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungu s. The ISME Journal, 3, 8 18 824. Hayes, M., Pounds, A., & Timmerman, W. 1989 . An Annotated List and Guide to the Amphibians and Reptiles of Monteverde, Costa Rica. Tyler, TX: The University of Texas at Tyler Print Shop. Krebs, C. 1989. Ecological Methodology. Ne w York, NY: Harper Collins Publishers. Kueneman, J., Wegener Parfrey , L., Woodhams, D., Archer, H., Knight, R., and McKenzie, V. 2014 The amphibian skin associated microbiota across species, space and life history stages . Molecular Ecology , 23 , 1238 1250 . Leenders , T. 2016. Amphibians of Costa Rica: A Field Guide. I thaca, NY: Cornell University Press. Longo, A., Savage, A., Hewson, I., and Zamudio, K. 2015. Seasonal and ontogenetic variation of skin microbial communities and relationships to natural disease dynamics in declining amphibians. R. Soc. Open Sci . 2: 140 377 . Longo, A. , and Zamudio, K. 2017. Environmental fluctuations and host skin bacteria shift survival advantage between frogs and their fungal pathogen . The ISME Journal, 11, 349 361 .
Microbial Diversity found on Anura Helmuth Malone 14 McKenzie, V.J, Bowers, R.M, Fierer, N., Knight, R., and Lauber, C.L. 2012 Co habiting amphibian species harbor unique skin bacterial communities in wild populations. The ISME Journal, 6, 588 596 . S.J . 2018 . Recent Asian origin of chytrid fungi causing global amphibian declines . Science, 360( 6389 ), 621 627 . Stebbins, R. & Cohen, N. 1995 . A Natural History of Amphibians. Princeton, NJ: Princeton University Press . Stone, D., Bowie, J., Tyler, M., and Wallace, J. 1992. The structure of caerin 1.1, a novel antibiotic peptide from Australian tree frogs . Journal of the Chemical Society , 17, 1224 1225. Walk e , J.B, et. al. 2014. Amphibian skin may select for rare environmental microbes . The ISME Journal, 8, 2207 2217 . Wargo, M., & Hogan, D. 2006. Fungal bacterial interactions: a mixed bag of mingling microbes. Current Opinion in Microbiology, 9 (4), 359 364. Woodhams, D. 2007. Symbiotic bacteria contribute to innate immune defenses of the threatened mountain yellow legged frog, Rana muscosa . Biological Conservation , 138(3 4 ), 390 398 . APPENDICES Appendix 1 : Frog Information Chart, containing species and common name, date, time, location, and place where frog was found, and total morphologies found on PTA (fungal) and TSA (bacterial).
Microbial Diversity found on Anura Helmuth Malone 15 # Species Common Name Date Time Location Place Fungal Bacterial 1 Pristimantis ridens Pygmy Rain Frog 14 June 2018 8:20 PM Santuario Leaf 14 N/A 2 Pristimantis ridens Pygmy Rain Frog 14 June 2018 8:45 PM Santuario Leaf 23 N/A 3 Rana forreri Dry Forest Leopard Frog 14 June 2018 9:00 PM Santuario Ground 14 N/A 4 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:00 PM Santuario Ground 17 8 5 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:01 PM Santuario Ground 17 13 6 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:03 PM Santuario Ground 15 8 7 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:05 PM Santuario Ground 16 10 8 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:10 PM Santuario Ground 10 9 9 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:15 PM Santuario Ground 11 5 10 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:25 PM Santuario Ground 12 9 11 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:30 PM Santuario Ground 9 8 12 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:40 PM Santuario Ground 11 10 13 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:40 PM Santuario Ground 11 6 14 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 7:42 PM Santuario Ground 9 7 15 Rana warszewitschii Brilliant Forest Frog 15 June 2018 8:00 PM Santuario Ground 7 10 16 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 8:06 PM Santuario Ground 10 9 17 Craugastor stejnegerianus Pacific Litter Frog 15 June 2018 8:20 PM Santuario Ground 9 10 18 Craugastor fitzingeri Common Rain Frog 17 June 2018 7:45 PM Bajo del Tigre Ground 17 10 19 Craugastor fitzingeri Common Rain Frog 17 June 2018 7:45 PM Bajo del Tigre Ground 9 10 20 Pristimantis ridens Pygmy Rain Frog 19 June 2018 7:30 PM Bajo del Tigre Leaf 4 7 21 Isthmohyla pseudopuma Meadow Tree Frog 22 June 2018 6:15 PM EstaciÃ³n Pond 8 11 22 Craugastor fitzingeri Common Rain Frog 22 June 2018 6:25 PM EstaciÃ³n Leaf 11 7 23 Isthmohyla pseudopuma Meadow Tree Frog 22 June 2018 6:30 PM EstaciÃ³n Pond 3 6 24 Isthmohyla pseudopuma Meadow Tree Frog 22 June 2018 6:45 PM EstaciÃ³n Pond 10 11 25 Pristimantis Pygmy Rain Frog 22 June 2018 6:55 PM EstaciÃ³n Leaf 3 12
Microbial Diversity found on Anura Helmuth Malone 16 Appendix 2 : Fungal Morphologie s Fungal Morphology Description Picture 1 White circle with blob edges 2 Yellow small circle 3 Cluster of dots without white space 4 White bubble circles, very round, clear 5 Egg looking collection of white circles 6 Dark grey circle ridens 26 Isthmohyla pseudopuma Meadow Tree Frog 22 June 2018 7:10 PM EstaciÃ³n Pond 2 8 27 Pristimantis ridens Pygmy Rain Frog 22 June 2018 7:35 PM EstaciÃ³n Leaf 3 9 28 Pristimantis cruentus Golden spotted Rain Frog 23 June 2018 6:40 PM Selvatura Leaf 6 4 29 Espadarana prosoblepon Emerald Glass Frog 23 June 2018 6:55 PM Selvatura Tree 8 8 30 Espadarana prosoblepon Emerald Glass Frog 23 June 2018 7:10 PM Selvatura Tree 8 6 31 Isthmohyla pseudopuma Meadow Tree Frog 23 June 2018 7:30 PM Selvatura Leaf 1 5 32 Pristimantis cruentus Golden spotted Rain Frog 23 June 2018 7:42 PM Selvatura Leaf 6 7 33 Diasporas hylaeformis Montane Dink Frog 23 June 2018 8:34 PM Selvatura Tree 5 7 34 Isthmohyla pseudopuma Meadow Tree Frog 23 June 2018 9:10 PM Selvatura Leaf 4 4
Microbial Diversity found on Anura Helmuth Malone 17 7 Flower looking grey collection of four circles 8 Aster spread with tendrils 9 Swirly intestinal blob 10 Yellow tendril circle 11 Grey circle within circle 12 Microphone blob 13 Grey four circle blob 14 Darker grey cloud 15 Clear white collection with soft tendrils 16 White collection with defined tendrils 17 Spindly tendril blob 18 White ghost blob
Microbial Diversity found on Anura Helmuth Malone 18 19 Pavement looking streak 20 Grey streak 21 Rust colored circle 22 Yellow ghost streak 23 Air pocket streak 24 Grey line streak 25 T shaped blob with bubbles 26 Yellow misshapen clear rectangle 27 Yellow/grey dark blob 28 Clear gooey stream 29 Grey with dark streaks blob 30 Yellow clustering of dots
Microbial Diversity found on Anura Helmuth Malone 19 31 White circle with clear inside 32 Yellow orange streak 33 White clear tendril blob 34 Triangle grey blob 35 More defined circular blob 36 White circle surrounded by clear circle 37 Grey misshapen blob 38 White round opaque circle 39 Yellow blob 40 Yellow orange blob 41 Octopus tendril blob 42 Yellow streak
Microbial Diversity found on Anura Helmuth Malone 20 43 Grey circle with orange inside 44 Grey circle with dot inside 45 Colorful blobs on top of each other 46 Suspended clear cloud growth 47 Grey circle with white inside 48 Jellyfish looking yellow blob 49 Grey snake blob 50 Organelle blob 51 Spaceship grey blob 52 Small black spot 53 Turtle looking blob 54 Blooming grey blob
Microbial Diversity found on Anura Helmuth Malone 21 55 Green hairy growth 56 Circle with black outline 57 Butterfly looking cirlce 58 Grey hairy growth 59 Egg looking yellow collection 60 Collection of dark blob surrounded by light blob 61 Insect looking blob 62 Green spot with white tendrils 63 Spattering of black spots 64 Heart shaped blob 65 Spread out streak 66 Pinwheel circle
Microbial Diversity found on Anura Helmuth Malone 22 67 Paint splatter blob 68 Grey fuzzy circle 69 Fuzzy boot blob 70 Astral projection 71 Double fuzzy blob 72 Three brown circles within each other 73 Pale white tendril, barely visible 74 Baby cloud with outline 75 Blob with yellow and black dots 76 Bead looking blobs 77 Multi yellow blobs 78 Yellow grey circle 79 Shell looking blob
Microbial Diversity found on Anura Helmuth Malone 23 80 Large white flower blob 81 Inhibition circle 82 Yellow flower with black outline 83 Streaks with lines 84 Brown ring circle Appendix 3 : Bacterial Morphologies Bacterial Morphology Description Picture A Yellow blob with dots on outside B Suspended cloud with green spot C Suspended dark triangle spot D Suspended dark circle spot E Fuzzy green spot F White atom ball G Spattering of black dots H Opaque goo blob
Microbial Diversity found on Anura Helmuth Malone 24 I Tendril blob with space J Rust orange double blob K Grey lava circle L Grey circle M Yellow circle N Double cloudy grey blob O Grey tiny circles P Flying birds Q Giant cloudy growth R Giant white cloudy growth S Grey streak T Ray looking yellow blob U Grey hairy blob V Grey blob with white spots W Green blob with clear ring X Yellow tendril blob Y Grey giant tendril blob
Microbial Diversity found on Anura Helmuth Malone 25 Z Grey defined blob AA Seahorse ridge blob AB Grey/yellow blob with dot AC Giant opaque cloud AD Tiny white dots AE Pale grey large blob AF Yellow blobs in a row AG White small tentacle blob AH Grey with orange middle blob AI Grey fuzzy outside blob AJ Spattering white blobs AK Yellow jellyfish blob AL Brown/yellow cloud AM Green/grey cloud AN Flower looking defined blob AO Scaly collection AP ER looking blob
Microbial Diversity found on Anura Helmuth Malone 26 AQ Mountain black outline, white circle AR Marshmallow blob AS Pale white cloud with dots AT Brown cloud with dots AU Sun circle AV White blob with outside tendrils AW Flying saucer AX Grey circle with middle dot AY Saturn blob AZ Saucer in cloud BA Head shaped blob BB Yellow brown circle BC Neuron blobs BD Blob with protrusions BE Pinwheel circle BF Grey circle within circle BG Bead streak line
Microbial Diversity found on Anura Helmuth Malone 27 BH Circle with outline BI Microphone line