3/11/2013 Microbial ecology of amphibian skin Ben Fitzpatrick Ecology & Evolutionary Biology University of Tennessee Microbial ecology of amphibian skin • The environment • Cutaneous biodiversity • Host defense and sseelleeccttiivviittyy • Conflict and cooperation 3/11/2013 Ben Fitzpatrick, University of Tennessee Microbial ecology of amphibian skin • The environment –Importance of skin in amphibian ecology –Climate –Skin structure –Skin secretions 3/11/2013 Ben Fitzpatrick, University of Tennessee 1 3/11/2013 Importance of skin in amphibian ecology • Respiration • Sensation • Absorb/release HO 2 • Heat exchange • Cocoon in drought • Chemical defense • Concealment/warning • Protection from abrasion & pathogens (Stebbins and Cohen 1995) 3/11/2013 Ben Fitzpatrick, University of Tennessee The environment: Climate • Amphibians have limited capacity to thermoregulate • Temperature and Humidityy – Warmer, wetter environments: More bacteria • pH – Might be most important environmental variable in global diversity 3/11/2013 Ben Fitzpatrick, University of Tennessee The environment: Skin structure animalbiology101.tumblr.com 3/11/2013 Ben Fitzpatrick, University of Tennessee 2 3/11/2013 The environment: Skin structure • Naked, permeable • Stratum corneum: Outermost layer • Sloughing continuous or episodic 3/11/2013 Ben Fitzpatrick, University of Tennessee The environment: Skin secretions • Mucus glands – Moisture, lubrication, mechanical trap – Mucins: gel‐forming glycoproteins • Granular glands – Pheromones – Defensive compounds: • Amines • Steroids • Alkaloids • Peptides http://vertebrateproject.wikispaces.com 3/11/2013 Ben Fitzpatrick, University of Tennessee The environment: Review • Skin a major interface in • Microbes have to contend amphibian ecology with • Constant contact with – Sloughing (disturbance) microbe‐rich media – Toxic secretions • EExxtteerrnnaall eennvviirroonnmmeenntt – Other microbes probablyinfluential • Microbes might gain – Abiotic – Enemy‐free space – Biotic – Nutritious secretions • Amphibian skin – Opportunity for infection structurally & chemically complex 3/11/2013 Ben Fitzpatrick, University of Tennessee 3 3/11/2013 Cutaneous biodiversity • Resident vs. transient microbes • Growth forms • AAbbuunnddaannccee • Diversity within and among individuals • Lauer et al. (2007)Copeia2007: 630‐640 3/11/2013 Ben Fitzpatrick, University of Tennessee Cutaneous biodiversity • Resident: – Grow/multiply on skin – Consistent abundance – Attached • Transient – Non‐reproducing – Exogenous – Lie free • Composition of samples in rinse water significantly • Lauer et al. (2007)Copeia2007: 630‐640 different from samples of remaining (attached) bacteria 3/11/2013 Ben Fitzpatrick, University of Tennessee Cutaneous biodiversity • Growth forms –Distinct rods, cocci –Smallmicrocolonies? –No evidence of biofilms • Lauer et al. (2007)Copeia2007: 630‐640 3/11/2013 Ben Fitzpatrick, University of Tennessee 4 3/11/2013 Cutaneous biodiversity • Abundance • Plethodon cinereus –Based on SEM (Lauer et al. 2007): –Average 17 million per cm2 –Fairly even distribution –Sometimes aggregated in/near glands 3/11/2013 Ben Fitzpatrick, University of Tennessee Cutaneous biodiversity • Abundance • Rhinellamarina –Based on cultivable plate counts (Meyer et al. 2012): –Average 0.5 to 2 million per cm2 –Depends on location (dorsal vs. ventral) and time since moult 3/11/2013 Ben Fitzpatrick, University of Tennessee Cutaneous biodiversity • Composition (16s RNA) Pond‐dwelling larvae (McKenzie et al. 2011) ‐100’s of OTU’s ‐Mostly ‐proteobacteria ‐Differences between salamanders (Ambystoma) and frogs (Rana, Pseudacris 3/11/2013 Ben Fitzpatrick, University of Tennessee 5 3/11/2013 Cutaneous biodiversity • Composition (16s RNA) Pond‐dwelling larvae Terrestrial (McKenzie et al. 2011) plethodontids(Allison ‐100’s of OTU’s 2012) ‐Mostly ‐proteobacteria ‐100’s of OTU’s ‐Differences between ‐Mostly ‐proteobacteria salamanders (Ambystoma) ‐No consistent differences and frogs (Rana, Pseudacris) among Plethonjordaniand Desmognathusocoee 3/11/2013 Ben Fitzpatrick, University of Tennessee Cutaneous biodiversity • Plethodontidsin GSMNP • Human (Roth & James 1988) – Nevskia() – Microccoci(Actinobacteria) – Hydrocarboniphaga() – Staphylococci (Firmicutes) – Koribacter(Acidobacteria) – Corynebacterium(Actinobacteria) – Burkholderia() – Propionibacteria(Actinobacteria) – PPseuddomonas(()) – AAciinettobbactter(()) • Pseudacristadpoles in AZ – Curvibacter() – Actinomycetales(Actinobacteria) – Sphingomonas() – Pseudomonadaceae() – Nevskia() 3/11/2013 Ben Fitzpatrick, University of Tennessee Cutaneous biodiversity • Terrestrial plethodontidsin 0P-4.teya-2halee0 05 SimGiSlaMr N“sPpecies” to other -4-2-20 P.jordani Axis 2-15-10-5 logs salamanders moist surfaces in the -0-4 D.ocoee -10 -5Axis 10 5 habitat -4-2 0 Rocks Different variants within -2 “species” -40 Logs 0 -2 -2 -4 -4 -4 -2 0-4 -2 0-4 -2 0-4 -2 0-4 -2 0 3/11/2013 Ben Fitzpatrick, University of Tennessee 6 3/11/2013 Cutaneous biodiversity • Terrestrial plethodontidsin GSMNP Similarity of microbial ccoommmmuunniittiieess oonn salamanders depends more on proximity than species I.e., interactions are diffuserather than specificor pairwise 3/11/2013 Ben Fitzpatrick, University of Tennessee Cutaneous biodiversity • Plethodontidsin GSMNP Salamander skin and cover objects have very ssiimmiillaarr bbaacctteerriiaall community structure. Specific strains or species differ between salamander skin and cover objects. 3/11/2013 Ben Fitzpatrick, University of Tennessee Cutaneous Biodiversity: Review • Distinct resident flora on • Diffuse rather then specific amphibian skin interactions • Millions per cm2: 10x to – Subtle differences between 100x that of human skin co‐occurring tadpoles and salamander larvae • Single cells or microcolonies – No differences between co‐ •• 110000’ss ooff OOTTUUss ooccccuurrrriinngg PPlleetthhooddoonn, • Composition very different Desmognathus from human skin • Community structure similar between • Composition different salamanders and cover between pond‐dwelling and objects terrestrial amphibians • Genetic differentiation between host‐associated and free‐living populations 3/11/2013 Ben Fitzpatrick, University of Tennessee 7 3/11/2013 Host defense and selectivity • Resistance and Containment • Antimicrobial Secretions • SSlloouugghhiinngg • Selectivity 3/11/2013 Ben Fitzpatrick, University of Tennessee Host defense and selectivity • Resistance and Containment –Stratum corneum –Mucus http://vertebrateproject.wikispaces.com 3/11/2013 Ben Fitzpatrick, University of Tennessee Host defense and selectivity • Resistance and Containment –Stratum corneum –Mucus • Antimicrobial Secretions http://vertebrateproject.wikispaces.com 3/11/2013 Ben Fitzpatrick, University of Tennessee 8 3/11/2013 Host defense and selectivity Antimicrobial Secretions • Amines –Histamines, epinephrine, adrenalin, bufotenins • Steroids –bbuffaddenolliddes–anesthhetics, carddiac stimullants • Alkaloids –100’s of compounds in poison dart frogs, sequestered from prey • Peptides –Likely most important antimicrobial compounds 3/11/2013 Ben Fitzpatrick, University of Tennessee Host defense and selectivity Sloughing (Meyer et al. 2012) • Disturbance/recolonization dynamics • May prevent “fouling” via biofilmformation • May increase vulnerability to opportunistic pathogens • Sloughed skin often eaten 3/11/2013 Ben Fitzpatrick, University of Tennessee Host defense and selectivity • Evidence of selectivity -4 -2 0 5 –Resident vs. transient 0P.teyahalee 0 –fSlaolraamanders vs. cover -4-2-20 P.jordani Axis 2-15-10-5 logs salamanders obbjjectts -4-0 D.ocoee -10 -5Axis 10 5 -2 -4 0 Rocks -2 -4 0 Logs 0 -2 -2 -4 -4 -4 -2 0-4 -2 0-4 -2 0-4 -2 0-4 -2 0 3/11/2013 Ben Fitzpatrick, University of Tennessee 9 3/11/2013 Host defense and selectivity • Salamanders vs. cover objects – Work in progress: Swabbed zigzag salamanders and cover objects GGeennoottyyppeedd PPsseeuuddoommoonnaass isolates Only one strain found on cover objects: P. putida 11 distinct strains found co‐ occurring on salamanders 7 related to a pathogen of fish 3/11/2013 Ben Fitzpatrick, University of Tennessee Host defense and selectivity • Salamanders vs. cover objects –Work in progress: Salamanders harbor a distinctand more diverse assemblage of Pseudomonas Do salamanders select a beneficial flora, or are they under attack? 3/11/2013 Ben Fitzpatrick, University of Tennessee Host Defense and Selectivity: Review • Barrier: Stratum corneum • Trap: Mucus • Poison: Antibiotic secretions • Removal: Sloughing Selection for certain microbial genotypes over others Prevention of competitive dominance? 3/11/2013 Ben Fitzpatrick, University of Tennessee 10
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