Does environment affect the diversity of microbes found on frog skin?


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Does environment affect the diversity of microbes found on frog skin?

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Does environment affect the diversity of microbes found on frog skin?
Translated Title:
¿Afecta el medioambiente la diversidad microbiana en la piel de las ranas?
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Yanelyn Perez
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Many frog populations are currently threatened due to a variety of reasons, but a prevalent reason is due to a fungal disease, chytridiomycosis, that attacks amphibian skin. Current studies suggest that possible resistance of chytrid fungus in certain frog populations can be attributed to the bacterial and fungal diversity found on the mucus of frog skin. In this study, I looked at whether various environmental factors can be correlated to a higher micro-biodiversity by swabbing individual frogs that were found in a variety of habitats. I swabbed 39 frog individuals in five different locations around Monteverde. I then grew fungal and bacterial colonies on agar plates to examine the biodiversity of the microbes on frog skin in respect to location, area which frog was found, and distance from water. My data shows that there was no significant difference between any of the environmental factors such as overall location of frog, the distance the frog was to the nearest source of water, and habitat that the frog was found on. The data did show significant differences in quantity of biodiversity of both bacteria and fungi in various species; and a significant increase for bacterial diversity in one species of frog that agrees with a past study. The data suggests that species may play a more prominent role in determining how much fungal and bacterial diversity is found on an individual rather than the location that the individual is in. ( , )
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Varias poblaciones de ranas se encuentran amenazadas debido a una variedad de factores, pero la predominante es la enfermedad fúngica quitridiomicosis, que afecta la piel de los anfibios. Se ha sugerido que la posible resistencia ante esta infección en ciertas poblaciones de ranas se relaciona a la diversidad de otros hongos y bacterias encontradas naturalmente en el mucus de la piel de las ranas. Evalué si factores medioambientales pueden correlacionarse con la micro diversidad de la piel de las ranas, recolectando isopados de ranas encontradas en distintos hábitats. Muestreé 39 ranas en cinco sitios de Monteverde. Luego cultivé hongos y bacterias en medios con agar para examinar la biodiversidad microbiana de cada rana en cada sitio, así como área en donde encontré a cada individuo. Mis datos muestran que no hay diferencias entre ninguno de los factores ambientales, distancia a cuerpos de agua, ni con el hábitat en donde encontré a cada rana. Mis datos muestran diferencias en la cantidad tanto de bacterias como hongos en varias especies. La especie de rana de la que se trate determina la diversidad de hongos y bacterias encontradas, y no tanto el sitio en el que ese individuo estaba.
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Student affiliation : Department of Ecology, Evolution, and Marine Biology; University of California Santa Barbara
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Microbial Diversity of Frog Skin Perez 1 Does environment affect the diversity of microbes found on frog skin? Yanelyn Perez Department of Ecology, Evolution, and Marine Biology University of California Santa Barbara EAP Tropical Biology and Conservation Spring 2019 7 th June 2019 ABSTRACT Many frog populations are currently threatened due to a variety of reasons, but a prevalent reason is due to a fungal disease, chytridiomycosis, that attacks amphibian skin. Current studies suggest that possible resistance of chytrid fungus in ce rtain frog pop ulations can be attributed to the bacterial and fungal diversity found on the mucus of frog skin. In this study, I looked at whether various environmental factors can be correlated to a higher micro biodiversity by swabbing individual frogs t hat were found in a variety of habitats. I swabbed 39 frog individuals in five different locations around Monteverde. I then grew fungal and bacterial colonies on agar plates to examine the biodiversity of the microbes on frog skin in respect to location, area which fro g was found, and distance from water. My data shows that there was no significant difference between any of the environmental factors such as overall location of frog, the distance the frog was to the nearest source of water, and habitat that the frog was found on. The data did show significant differences in quantity of biodiversity of both bacteria and fungi in various species; and a significant increase for bacterial diversity in one species of frog that agrees with a past study . The data suggests that s pecies may play a more prominent role in determining how much fungal and bacterial diversity is found on an individual rather than the location that the individual is in . ¿ Afecta e l medioambiente la diversidad microbiana en la piel de las ranas? RESUMEN Varias poblaciones de ranas se encuentran amenazadas debido a una variedad de factores, pero la predominante es la enfermedad fúngica quitridiomicosis, que afecta la piel de los anfibios. Se ha sugerido que la posible resistencia ante esta infección en c ie rtas poblaciones de ranas se relaciona a la diversidad de otros hongos y bacterias encontradas naturalmente en el mucus de la piel de las ranas. Evalué si factores medioambientales pueden correlacionarse con la micro diversidad de la piel de las ranas, r ec olectando isopados de ranas encontradas en distintos hábitats. Muestreé 39 ranas en cinco sitios de Monteverde. Luego cultivé hongos y bacterias en medios con agar para examinar la biodiversidad microbiana de cada rana en cada sitio, así como área en don de encontré a cada individuo. Mis datos muestran que no hay diferencias entre ninguno de lo s factores ambientales, distancia a cuerpos de agua, ni con el hábitat en donde encontré a cada rana . Mis datos muestran diferencias en la cantidad tanto de bacteria s como hongos en varias especies. L a especie de rana de la que se trate determina la diversidad de hongos y bacterias encontradas , y no tanto el sitio en el que ese individuo estaba. _______________________________________ __________________________________ _____

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Microbial Diversity of Frog Skin Perez 2 It is crucial to understand various aspects of amphibian species such as physiology, ecological relationships, and populations dynamics because the Anuran group is suffering from population declines and extinctions since the 1980s. (Beebee & Griffit h 2005.) Pollution, introduction of invasive species, climate change, fungal diseases, and viral diseases are agents that trigger massive population declines. (Beebee & Griffith 2005.) However, the fungal disea se, chytridiomycosis ( caused by Batrachochytri um dendrobatidis or Bd), is theorized to be the main cause of the rapid mortality rate in Anuran groups (Berger et al. 1997 & Kilpatrick et al. 2010.) Recent studies, however, found that various frog populations in Panama have started to recover after bein g near extinct d ue to Bd outbreaks (DiRenzo et al. 2018) and that various frog populations have shown resistance to the effects of the fungus (Retallick et al. 2004.) In some studies, resistance is correlated with a greater diversity of the microbiota, a c ommunity of bact is a fungus, most fungi and bacteria living in a microbiota found on a host organism can have mutualistic relationships; some bacteria have been sho wn to have antif ungal and antibacterial properties that can prevent infection or harmful pathogens from establishing colonies on the host organism (Brucker et al. 2008.) Cutaneous respiration is the process of respiration using skin rather than lungs or g ills (Duellman & Trueb 1994.) All frogs use this as a secondary form of respiration, usually when submerged under substrate or water (Duellman & Trueb 1994.) For frogs to remain healthy, their skin must maintain proper moisture and a balance of ions to su pport the cutane ous respiration (Duellman & Trueb 1994.) Amphibian skin achieves this by creating a layer of mucus secretions that is constantly on the skin; these conditions are also ideal for fungal and bacterial growth (Duellman & Trueb 1994.) The micro biota found on f rog skin has become an increasingly studied aspect of frog populations because it may be a factor that can help indicate the likelihood of populations surviving a chytridiomycosis outbreak (Brucker et al. 2008 & Woodhams et al. 2007. ) By un derstanding whic h factors play a role in the selection of microbial diversity in certain groups, researchers will have a better grasp of which external conditions determine if certain populations are susceptible to outbreaks. S uch knowledge is crucial as a mphibian populat ions are declining and any insight into possible solutions is greatly needed. Because of this, I am interested in looking at whether environmental factors can help determine the quantity of bacteria and fungi found on frog skin. Due to th e high abundanc e of frog species occurring in Costa Rica, there are various frog species that can be found locally and can inhabit a large range of habitats that include aquatic, fossorial, and aboral. I hypothesize that different locations, habitats, and distance from w ater will be significant in determining whether a frog has higher microbial diversity. I predict that frogs that are normally found near water are more likely to have a higher bacterial and fungal diversity on their skin. I also predict that frogs that are found in ponds are more likely to have higher microbial diversity. I believe these tw o occurrences will happen because most frog species that are near water have a reservoir of bacteria and fungi that they musts constantly must interact wit h. The farther away a frog is found from a water source, it can be implied that these frogs are speci es that may not need to be near a body of water to keep their skin hydrated but by other means such as rain, rehydrating habits, or using bromeliads (Duell man & Trueb 1994.) I also predict that frog s found in locations that have more human disturbance sho uld yield higher diversity as pollutants or fertilizers can

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Microbial Diversity of Frog Skin Perez 3 increase microbial yield in an area. In two previous EAP projects, both completed within the pa st year, there i s evidence to suggest that species type and location are significant aspects in deter mining the biodiversity of microbes on frog skin (Beltran 2018 & Helmuth Malone 2018.) My study focuse d on distinct locations with diverse environmental co nditions and examined the relationship between location, habitat type distance from water, and specie s type. MATERIALS AND METHODS Field Methods Over the span of ten nights, I visited five different locations across Monteverde and took swab samples from thirty nine individuals. These locations had a body of water (such as a creek or pond) on the property and were accessible by trail. These surveys occurred between the hours of 6pm 10pm and only occurred during favorable weather conditions; it was too dang erous to be outside during lighting storms and to be in certain areas during heavy rainfalls due to a previous history of landslides. I visited the following locations: Estaci ó n Biol ó gica, Monteverde Institute, Santuario Ecol ó gico, L ife Monteverde farm, an d San Gerardo Biological S tation . With the help of Felix Salizar and Eladio Cruz, I identified the genus and species of frogs. Wearing clean gloves and using freshly opened cott on swabs, I swabbed each individual frog thirty times with two swabs: ten times on the dorsal side, ten times on the ventral side, and five times on each inner side. After swabbing and identifying the species, the individual was released back into its habi tat. To minimize spreading pathogens from frog to frog, I wore a new pair of gl oves per individual and discarded contaminated gloves into a bag. I kept swab samples in individual ziplock bags to minimize contamination and labeled it accordingly. The glove s, Ziploc bags, and swabs were all thrown away in a bigger ziplock bag then dis carded back at the institute. I also where the frog was found on (grass,leaves , pond, tree, or ground.) Laboratory Methods: Prior to starting my fieldwork in various locations of the Monteverde area, I prepared agar plates in the laboratory of the Monteverde Institute. I used a total of thirty three petri dishes and a pressure coo ker to create the plates. I used two different types of plates: Potato Dextrose Agar (PDA) for fungal growth and Tryptone Soy Agar (TSA) for bacteria growth; these methods are based on the paper produced by EAP student project, Lilly Helmuth Malone. I used the recipe as followed: 16 grams of TSA powder added onto 400mL of distilled w ater in an autoclavable 500ml glass bottle with a lid and 15.8 grams of PDA powder added to 400mL of distilled water in an autoclavable 500ml glass bottle with a lid. Using the pressure cooker, I sterilized glass plates and agar solutions by adding 500mL o f water to the bottom of the pot and applying approximately 20 minutes of constant heat. Before sterilizing the glass petri dishes, I washed them with dish soap and water and le t them dry; when adding the plates to the pressure cooker, I used a metal stand to prevent the plates from touching the metal and water on the bottom. I added approximately 10mL of agar per plate and waited about an hour for it to cool down before sealing it with parafilm and placing it in the refrigerator. Once I had collected the swabs from individual frogs, I headed back to the Monteverde Institute within two hours of collecting the swabs and streaked agar plates with the samples. I followed this p rocedure for all but two nights due to time and materials conflicts: May 20 th and

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Microbial Diversity of Frog Skin Perez 4 Ma y 21 st, where I streaked samples within 12 hours of collection and placed them in a refrigerator or cooler prior to streaking. I used a swab for the dorsal side and one used for the ventral side per each sample. When I streaked TSA and PDA agar plates, I used a zig zag motion across the plate and swabbed half a plate with the dorsal swab and the other half with ventral because I was examining the micro biodiversity of the frog individual overall. I used two agar plates to test bacterial and fungi growth fo r each individual. I sealed the streaked plates with parafilm and placed them (agar side on top) into an incubator, which was set at around twenty one degrees Celsius for a minimum of forty eight hours. After forty eight hours, I looked at the growths on the p lates and attempted to identify them based on colony morphology using the dissecting scope. I classified the fungi and bacteria based on different morphologies of colonies using shape, color, texture, and the plates used; I classified fungal growth using PDA plates and bacterial growth using TSA plates. I created a n appendix for both types of plates with descriptions of my morphospecies and pictures. To count microbial diversity, I counted morphospecies and number of colonies I observed on both TSA a nd PDA plate s. I decided to divide the number of morphospecies by the number of colonies because I saw that some plates grew an abundance of colonies but with only few morphospecies while other plates grew fewer colonies but had a greater abundance of diff erent morphospecies; by dividing the number of morphospecies by colonies, I c an account for these differences. I followed the waste procedure that was used in the Helmuth Malone (2018) project. After I finished using the agar pl ates, I placed them in a bu cket o f 20% bleach for 24 hours to sterilize any microbial growth. I then washed the glass Petri dishes with soap and water and either reused them or put them away in the appropriate box. Data Analysis Used: The data I collected for this study equal variances in the population and relied more on categorical results than numerical results . As a result, I used the Kruskal Wallis test, a nonparametric ANOVA, to check for differences in ranks between location, area, and species . I also used the Kru s ka l Wallis test to compare the differences in microbial diversity in I.psuedopuma , to account for differences in species in respect to the location s where species was found. To test for difference in means between groups, I used a t test followed by the r e sults in the Kruskal Wallis test. I used a linear regression to test for significance in distance from water to higher microbial diversity as this relationship will show me if higher diversity is correlated to being near water. RESULTS For this study, I looked at thirty nine individuals of ten different species at five different locations. The most specious location was San Gerado and the least were Institute and Estacion (Table 1.) For PDA plates, I counted forty two different morphospecies (Appendix 1 ) . For TSA plates, I counted thirty one different morphospecies (Appendix 2 ) . The factors that I looked at were: overall location of survey, area in which individual was found, distance of where individual was found to closest water source, and species. I took these factors and compared them to both the biodiversity values I calculated for PDA plates and the TSA plates. For this

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Microbial Diversity of Frog Skin Perez 5 section, both fungal and bacterial diversity were tested separately but if they show similar trends, I will refer to i When looking at locations, I saw that there were no significant overall differences between all locations when using at Kruskal Wallis test ( p value>0.05 ) and no significant overall differences in mea ns when using a student t test (p value >0.05). In terms of species, there were no significant overall differences between all species when using at Kruskal Wallis test (p value>0.05.) Using the student t test to look at overall differences between means, two species had a significant difference in bacterial diversity, I.psuedopuma (p value =0.032) and E.prosoblepon (p value =0.020) ; two species also had a significant difference in fungal diversity, L.taylori (p value 0.022) and C.fitzengeri (p value = 0.018.) When testin g for the differences in diversity for I.psuedpuma in the two locations it was found, there was no significant ove rall difference between the two location using the Kruskal Wallis test and student t test. When testing the difference between different habit at types, there were no significant overall differences when using at Kruskal Wallis test (p value>0.05) and no si gnificant overall differences in means when using a student t test (p value > 0.05 .) Using the linear regression model to look at the correlat ion between distance of water and diversity, both TSA and PDA showed very low R squared values (TSA = 0.0083 and P DA= 0.0566) and were not significant (p value> 0.05) LOCATION NUMBER OF SPECIES SEEN SPECIES NAME Estacion 2 Isthmohyla pseudopuma, Lithoba tes taylori Institute 2 Craugaster fitzingeri, Espadarana prosoblepon LIFE 4 Diasporus diastema,Lithobates warszewitschii, Pristimantis ridens, Isthmohyla pseudopuma San Gerado 6 Craugaster crassidigitus, Craugaster fitzingeri, Craugaster underwoodii, D uellmanohyla rufioculis, Espadarana prosoblepon, Pristimantis ridens Santuario 3 Lithobates warszewitschii, Lithobates taylori, Pristimantis ridens Table 1 N umber of species found in every location visited in the study and the type of species that were seen. San Gerardo had the most amount of species (6) while Estacion and Institute had the lowest amount (2). .

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Microbial Diversity of Frog Skin Perez 6 LOCATION BRIEF DESCRIPTION Estacion Biologico Higher altitude, human made ponds , creeks, moderately p rotected area Monteverde Institute High altitude, natural creek far in the reserve, near a farm, public hiking trail Santuario Ecologico Low er altitude, human made ponds, private property San Gerado Field Station Lowest altitude, abundance of natural c r eeks, very protected area LIFE Monteverde Low altitude, natural creek, area around trail is a farm 0 0,05 0,1 0,15 0,2 0,25 0,3 Estacion Institute LIFE trail Santuario Ecologico San Gerado Average # of Morphos per Colonies Location of Survey TSA PDA Figure 1 A ver ages of bacterial and fungal based on the overall location in which individuals were found. This data was tested for significance using Kruskal Wallis test and chi squared approximation. There are no significan t differenc es between the group s (p>0.05) . Alt hough there is no difference between all groups, Estacion shows a slightly increase in the average for bacterial diversity while the institute average shows a slight decrease . in fungal diversity. Table 2 D ifferences between the five locations used during this study. San Gerardo was the least disturbed by human activity as it is a reserve while LIFE Monteverde is the most disturbed since it is a farm

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Microbial Diversity of Frog Skin Perez 7 Figure 2 B acterial and fungal diversity averages according to species. There seems to be little differences, but I.psu edopuma seems to show a higher bacterial diversity compared to other groups. Based on the Kruskal Wallis test and chi squared p value, there is no significant difference between species overall. However, when looking at specific differences in mean of grou ps using student t tests , two species show significant differences in TSA diversity (p value= 0.035 for I.psu edopuma and p value=0.020 for E.prsoblepon ) and two species show significant differences in PDA diversity (p value =0.018 for C.fitzingeri and p va lue = 0.022 for L.taylori ). 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 Average # of Morphos per Colonies Species TSA PDA 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 Estacion San Gerado Average # of Morphos per Colonies I.psuedopuma TSA PDA Figure 3 A verages in I.psuedopuma , a species found in two of the five locations, to see trends in microbial diversity in accor dance to location. This figure shows that there are no differences in averages in overall biodiversity between locations. The Kruskal Wallis test and student t test also confirmed that there are no significant overall differences (p value >0.05)

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Microbial Diversity of Frog Skin Perez 8 0 0,05 0,1 0,15 0,2 0,25 0,3 grass ground leaf pond tree Average # of Morphos per Colonies Area Individual was Found On TSA PDA R² = 0,0566 0 0,1 0,2 0,3 0,4 0,5 0,6 0 20 40 60 80 100 Distance From Water (meters) R² = 0,0083 0 0,2 0,4 0,6 0,8 1 1,2 0 20 40 60 80 100 Distance From Water (meters ) Figure 4 A verages in areas in which the individuals were fo und on such as thee grass, ground, leaf, pond, and tree. Based on these results, there seems to be a lower average in bacterial diversity in individuals found on trees and a lower average in fungal diversity in individuals found on leaves. When looking at the Kruskal Wallis test and student t test, there are no significant overall differences. Fi gure 5 R elationship between the diversity of fungi on an individual versus the distance of where the individual was found to the nearest source of water. As shown in the graph, there is no significant correlation between the two factors and can b e prov ed by the small R squared value. Figure 5 R elationship bet ween the diversity of bacteria on an individual versus the distance of where the individual was found to the nearest source of water. As shown in the graph, there is no significant correlation between the two factors and can be proved by the small R square d value.

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Microbial Diversity of Frog Skin Perez 9 DISCUSSION Overall, most of the factors that I decided to look at in this study were not proven to be significantly different from one another. Thus, my hypothesis that environmental factors are important in d etermining the quantity of microbial biodiversity was not supported in this study as there was no difference in locations (Figure 1), no difference in habitat (Figure 4), and no difference in distance from water (Figure 5 and 6.) My predictions that higher microbial diversity would exist in individuals that live on ponds and near water were not proven (Figure 4,5, and 6 .) My prediction that frogs found in locations that have more human disturbance have higher overall diversity was also not proven (Figure 1. ) However, there was one significant relationship, and it was between species and quantity of micro biodiversity (Figure 2 .) A possible explanation for this trend could possibly be that species type is more likely to play a role in determining the quantity of biodiversity in the skin of frogs than location . It can be concluded that the reason the Estacion had a higher bacterial diversity average of all the locations (Figure 1) was because of I. p suedopuma was one of the two species seen in the Estaci ó n (Tab le 1) and ha d a significant difference and a higher bacterial diversity average among all the other species (Figure 2 .) The idea of species being important can be furthered enhance d by the other three species that showed significant differences in both bac terial and fungal diversity. No other factor showed significant differences among the groups. The locations that were used throughout the study all had different characteristics compared to each other (Table 2 ) and was evident by the species occurrence in them (Table 1) . This is can be observed by the diverse species found in San Gerado and the lack of species found in the Estacion and Institute. Looking at Figure 3, it is evident that despite being in different locations, I.psuedopuma ha d no significant d ifferences in overall micro biodiversity in regards to location. Referring to Figure 1, there is also no significant differences in overall locations, thus strengt hening the statement that environmental factors are not important in determining the quantity of microbial diversity found on frog skin. When looking at previous EAP studies (Beltran 20 18 & Helmuth Malone 2018), it was seen that species can play a role i n determining the number of overall microbes found in the skin. T he Helmuth Malone article als o saw that I.psuedopuma had a higher bacterial diversity, which is similar to what happened in this study (Figure 2) . However, that study also saw that P.ridens had the highest fungal diversity, as well as an increase in fungal diversity in C . fitzengeri a nd C . stejnegerianus . Helmuth Malone (2018) attributed this trend to the area that the frogs were found, such as leav es , ponds, and ground . H owever, my study did not have data to support this claim (Figure 4). When looking in the Beltran article (2018) , the re was also a claim that P.ridens had the highest fungal diversity, which was not supported by my study as well (Figure 2 ). Instead, my study suggests that L.taylori had a significant difference and Figure 2 implies that is has increase of fungal diversity and C.fitzengeri had a significa nt difference and Figure 2 implies that it has a decrease in fungal diversity. Differences between this study and the previous two studies could be traced back to weather, sample size, and methods. Both Helmuth Malone and Beltran studies occurred in 2018,

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Microbial Diversity of Frog Skin Perez 10 this year has had different weather patterns compared to last year, which can possibly attribute to the variances in microbes and frog species found. In addition to that, Beltran performed her study in the Fall, which saw a more wet season and o ppor tunities to see frogs as they prefer wet conditions. This study also looked at the Institute and San Gerado as locations, wh ile the other two studies did not. Beltran had more frog individuals (n=55) than this study(n=39) and He lmuth Malone (n=34) . This st udy saw ten species of frogs, meanwhile, Beltran saw eight and Helmuth Malone saw nine ; all three studies had few species that were unique to their studies. In addition to this, my study used a lower temperature in the incubator (21 degree Celsius) versus the other two studies that used over 30 degrees Celsius ; I chose a lower temperature because of experience with fungal and bacterial growth in a laboratory setting , which could affect the species and colonies that were formed in both studies. This study al so autoclaved the agar solution , which the other two studies did not do as well, as it is a technique to ensure that the agar solution is also sterile . This difference in preparing the agar could have varying affects on the grow th of bacteria that I could not foresee . Possible areas of errors include possible contamination in preparing material as there is little aseptic tools in the laboratory at Monteverde, possible contamination during swabbing and packaging of the samples, a nd the delay of tw elve hours for handling some samples. These factors can possibly be a reason why I did not get results that fully resembled the last two studies. If contamination occurred, it could skew the results of my study without notice. Having cont rol plates would be beneficial for future studies to account for contamination. Since this study shows that species may play a prominent role in determinin g whether a population has a higher biodiversity, this can suggest that the micr o biome found on the skin of frogs can somewhat be regulated by the frog species. A possible explanation for this can be attributed to different frog adaptations that are needed for various environments. Frog species, depending on their habitat, can have variable textures and thickness of skin. (Duellman & Trueb 1994 .) As there are some species that have different skin compositions and rehydrating adaptations to survive in habita ts that have less moisture , this can imply that this rehydration adaptations and sloughing (shedding of outer amphibian skin layer) can possibly play a role in maintenance of micr o biomes. I theorize this may play a role in the amount of microbial biodivers ity that can be found on frog skin as some species may be adapted to slough more frequently than oth ers, thus shedding their skin and microbes often (Duellman & Trueb 1994 .) I also theorize that the various adaptations to rehydrate skin among species may play a role in determining how much biodiversity is found on skin (Duellman & Trueb 1994 .) Future s tudies can possibly compare methods found in my study and previous studies to see if there really is a relationship or if patterns are just random . Perhaps a future study can look at one species of frog that occurs in various locations and see if the quant ity of the microbial diversity changes per location to see how important species is in determining that. Perhaps a study can also look at the uniqueness of microbial diversity per individual instead of looking at the quantity of microbial diversity; doing this can imply that some species of frogs or environmental factors can contribute to uncommon combination of microbes. This can show that environmental factors or sp ecies can possibly determine what kinds of microbes can be found on the frog of skin. Overa ll, further research on this subject can help further current understanding of the relationship

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Microbial Diversity of Frog Skin Perez 11 that frogs have with these microbes, how they acquire these microbes, and what factors could dictate what types of microorganisms one can find on the skin of fr ogs. AKNOWLEDGEMENTS I would like to thank Federico Chinchilla for being an amazing advisor and helping me figure out what to do while still allowing me to be indep endent throughout this whole process; and thanks to Frank Joyce for being my second reade r, giving me valuable advice, and allowing us to visit San Gera r do during the study. I would also like to thank Felix Salizar for helping me learn about frog species a nd giving advice on how to find frogs. I would especially like to thank fellow EAP studen t, Elizabeth McDonald, as we both worked together to find frogs and spent countless nights clicking our tongues in the forest together. I would also like to thank Elad io Cruz , Christopher Sal a zar, Jerson Santamaria for helping us out and giving us their ex pertise. A big thanks to Monteverde Institute for allowing me to use their lab and trail during late nights and to Luisa Moreno, who helped me figure out the whole lab oratory situation. Another thanks to Estaci ó n Biol ó gica, LIFE Monteverde, Santuario Ecol ó gico, and San Gerado Biological Reserve for allowing us to visit. Finally, thanks to all the frogs that were really cute and that let me catch and swab them! LITERAT URE CITED Beebee, T . J . C . , and R . A. Griffiths. 2005. The amphibian decline crisis: a watershed for conservation biology? Biological C onservation 125 ( 3 ) : 271 285. Beltran, A . 2018 Fungal morphs in Lithobates warszewitschii and other frogs . EAP Fall 2018 , Course Book . Berger, L . , R . Speare, P . Daszak, D. E . Green, A . A. Cunningham, C. L . G oggin, R . Slocombe . 1998. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Au stralia and Central America. Proceedings of the National Academy of Sciences 95 ( 15 ): 9031 9036. Brucker, R. M., Harris, R. N., Schwantes, C. R., Gallaher, T. N., Flaherty, D. C., Lam, B. A., & Minbiole, K. P . 2008. Amphibian chemical defense: antifungal metabolites of the microsymbiont Janthinobacterium lividum on the salamander Plethodon cinereus . Journal of C hemical E cology . 34(11) : 1422 1429. DiRenzo, G . V., et al. 2018. Eco evolutionary rescue promotes host pathogen coexistence. Ecologi cal Applications 28 ( 8 ) : 1948 1962.

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Microbial Diversity of Frog Skin Perez 12 Duellman, W . E., and L . Trueb . 1994 . Biology of Amphibians. Johns Hopkins University Press . Helmuth Malo ne, L . Microbial diversity found on a nura in the Monteverde area . EAP Spring 2018 Course Book. Kilpatrick, A. M . , C . J. Briggs, and P . Daszak. 2010. The ecology and impact of chytridiomycosis: an emerging disease of amphibians. Trends in Ecology and Evoluti on . 25 ( 2 ): 109 118. Retallick, R ., W . R . H . McCallum, and R . Speare. 2004. Endemic infection of the amphibian chytrid fungus in a frog community post decline. P l oS B iology 2 ( 11 ): 351. Woodhams, D . C., et al. 2007. Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses. Animal Conservat ion 10 ( 4 ): 409 417 APPENDIX Appendix 1 shows the morphospecies list I crea ted for Tryptone Soy Agar which accounted for Bacterial growth NAME DESCRIPTION # OF SAMPLES SEEN IN Fuzz Grey, hyphae, single colonies, "hairy" 10 Swirl white, thick hair like filament, then swirl with other filmanets branching out 6 Slimey transparent, sightly yellow, leaf like spread pattern 10 White Blob Round colonies, white, opaque, very bolbous 11 Yellow Blob Round colonies, dark yellow, opaque, very bolbous 1 1 Flower Blob round colonies, light yellow transparent on edges, opaque yellow in middle 3

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Microbial Diversity of Frog Skin Perez 13 Ghost very watery looking, slimely , some hard bodies that are whte and trapsarent 24 Yellow ghost watery, small hard yellow body 8 Black Dot small black dots 1 Coke Small, dark, white bodies 2 Smear very transparent, slightly yellow 15 FuzzSwirl swirly, hair like filament, branching, fuzzy center 2 Yellow Egg flowerly edge that is white, transparent, hard yellow center 2 Bush hyphae fuzzy filaments, fuzzy rou nd, green brown center 8 frosty filaments, looks frosty 4 pasty light yellow blobs, not round, just very light yellow 3 Blood wave bright red, not round, wave/leaf like pattern 1 Soft white blob white blobs that are very trnaslucent 2 teal Bush Teal c enter, fuzzy filements that branch out , round, some white 2 red fuzz super dark base with white fuzz growing out of it 1 Hardy Yellow Mustard dark yellow spread thinly -can have fuzz grow on it 2 Pink ghost pink looking, watery body 1 Leafy Vienatio n, waves out, very filamous in the colony, white 4 Geopgraphy wave like pattern, textured, yellowish color, a big blob 2 transparent leaf white, almost tranpsarent, big bodies that have leaf spread with slight outline 4 Piss yellow bright yellow, tranps arent, blob like 3 white smear pale, whiteish, smear pattern that isn't very translucent 3 soft edge pale white body, transparent, edges are feathered out and are more translucent then center 2

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Microbial Diversity of Frog Skin Perez 14 branch WHITE, dark brown, strand like colonies, branching o ut 1 yellow brown bush hyphae fuzzy filaments, fuzzy round, brown then yellow center 1 egg sac transparent white blobb with filements inside it, it looks like a squishy egg sac 1 Appendix 2 shows the morphospecies list I created for Potato Dextrose Agar which accounted for Fungal growth NAME DESCRIPTION # OF SAMPLES SEEN IN Wo rmy white, creamy, transparentish, colony with strands of hair in "center" 1 Fuze fuzzy, no clear center, filements that are short and everywhere, 17 Eye Fuze fuzzy, pink/reddish center, filements that are short and everywhere, round and spread 1 EyeBa ll round white center, with black dot at center, has filemnts coming out of it, not necessarily round 1 Squigly Round edge, clear center, small squigly lines in middle 5 white blobs round edge, white, not transparent 15 Bush dark brown, green center, fu zzy fements that branch out, round, some white 21 yellow head round, transparent edege of circle, yellow, opaque center, circle within a circle 8 wave white, transparentis, wazy pattern from filament, roundish 3 smear very transparent, slightly yellow 1 3 pasty light yellow blobs, not round, just very light yellow 10 teal Bush Teal center, fuzzy filements that branch out , round, some white 10 Wave fuzz fuzzy filaments that branch out like waves, clear, white 11 yellow wave clear, transparent center, yellow edges 4 Green Fuzz vibrant green filaments that extend outwards, no white 3

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Microbial Diversity of Frog Skin Perez 15 flower fuzz Yellow transulcent center, fuzzy filements that branch out 3 yellow blob watery, small hard yellow body 11 Swirl white, thick hair like filament, then swirl with other filmanets branching out 3 Coke white, tiny dots, all compact together 2 Yellow fuzz hard yellow, 3d, fuzzy filaments that spread out 8 Ghost very watery looking, slimely, some hard bodies that are whte and trapsarent 16 Peach fuzz peach col ored, 3d, fuzzy filaments that spread out 8 Squigly Yellow yellow blob, with hard yellow inside, and squigly lines covering it all 2 Yellow Ghost watery, small hard yellow body 12 red fuzz red base with white fuzz growing out of it 4 Snowball slightly fuzzy, mostly all white, round 13 green dots small green dots, scattered pattern 3 White head transluscent outer edge, white hard center 4 Yellow blob hard yellow, super duper dark yellow, round 2 brown flower frost round, outer edge looks flowered ou t,white inner edge, brown outer edge, ring in center 2 Yellow plastic yellow blob, with light yellow base, and curled up darker yellow regions 1 frosty filaments, looks frosty 7 Hardy Yellow Mustard dark yellow spread thinly -can have fuzz grow on it 7 bubble bubble structure that looks wet -not quite round bt protuding from agar 1 red ghost watery, pink/redish 1 rust rust color, spreads, yellow outline, flat 1 brown frost somewhat fuzzy, brown frost patterns, white around 3 transparent leaf whit e, almost tranpsarent, big bodies that have leaf spread with slight outline 1 eye bush dark brown, green center, fuzzy fements that branch out, round, white in the center look like an eye 2 yellow spread yellow color, not squishly , little dots, looks lik e typical slime moldish 1

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Microbial Diversity of Frog Skin Perez 16 Rust fuzz super rust/brown red base with white fuzz growing out of it 1 Outliney weirdly shape blob with a hard whte outline, wavy inside 2


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