2012, 第 17 屆細菌學研討會 (8/29-8/31, 溪頭)
- Chang YH, Liu W, Fang TT, Lin SY, Dai FW, Huang WC, Yang YC, Chen CY. 2012. Gut microbiota of an arboreal green snail Leptopoma nitidum taivanum.
- Wang YN, Chen CY, Chang SC. 2012. Microbial diversity in soil and bryophytes at Chi-Lan Mountain yellow cypress forest.
- Chen TY, Chen CY. 2012. Microbiota associated with rhodophyte Galaxaura filamentosa in Hualien.
- Hou PY, Kuo YH, Chang SC, Chou PH, Chen CY. 2012. Islands in an acid sea: bacteria living on plants growing on acidic grounds.
- Wan MH, Chen YC, Chen CY. 2012. Microbial diversity associated with colonial Palythoaanemones in Hualien.
- Chang YH, Chen CY. 2012. Environment is a major factor determining the composition of Polypedates braueri tadpole gut microbiota.
- Hsu YT, Chang YH, Chen CY, Yang YC, Ho HC. 2012. Diversity in gut microbiota ofKurixalus eiffingeri tadpoles.
- Chen H, Chen WC, Chen CY. 2012. Will the Vibrio vulnificus mutants finally take over the sea?
Gut microbiota of an arboreal green snail Leptopoma nitidum taivanum
Yung-Hsin Chang (張永欣)1, Wei Liu (劉薇)2, Tzu-Ting Fang (方姿婷)2, Sin-Yin Lin (林昕穎)2, Fei-Wu Dai (戴非無)2, Wen-Chin Huang (黃文琴)2, Yu-Chang Yang (楊育昌)3, Chun-Yao Chen (陳俊堯)1
Department of Life Science, Tzu-Chi University, Hualien1
Hualien Girls Senior High School, Hualien2
Endemic Species Research Institute, Council of Agriculture, R.O.C., Nantou3
Gut microbiota can directly affect host health. Symbiotic gut microbes may digest or synthesize nutrients that cannot be acquired by their animal hosts. Leptopoma nitidum taivanum is a green snail species which has a wide distribution in Taiwan. This species is mostly arboreal, however it is not clear whether it is saprophagus or phyllophagus. We examined individuals collected in Yilan and Hualien in eastern Taiwan. DGGE analysis indicated that gut microbiota of individuals collected in the same sampling site or different site are quite similar. The gut microbial community has low species richness and evenness, judging from the DGGE profiles. We found high percentage of cellulolytic organisms in bacterial culture from gut homogenate. Bacillus cereus, B. megaterium, B. thuringiensis, Microbacterium aurum, Pseudomonas plecoglossicida, and Aeromonas hydrophila were isolated from direct plating of gut homogenate. Stenotrophomonas maltophilia and M. binotii were obtained after filter paper enrichment procedure. These cellulolytic gut microbes may help the snail in degrading plant-derived cellulose-rich food. During 5 field surveys in Taroko National Park, highest occurrence of L. nitidum taivanum was seen on Arenga engleri (23%), but they are even more frequently found on off-tree non-living materials (46%). From dissecting observation, low diversity of gut microbiota, and high occurrence on off-tree locations, we hypothesize that L. nitidum taivanum is mainly saprophagous.
Department of Life Science, Tzu-Chi University, Hualien1
Hualien Girls Senior High School, Hualien2
Endemic Species Research Institute, Council of Agriculture, R.O.C., Nantou3
Gut microbiota can directly affect host health. Symbiotic gut microbes may digest or synthesize nutrients that cannot be acquired by their animal hosts. Leptopoma nitidum taivanum is a green snail species which has a wide distribution in Taiwan. This species is mostly arboreal, however it is not clear whether it is saprophagus or phyllophagus. We examined individuals collected in Yilan and Hualien in eastern Taiwan. DGGE analysis indicated that gut microbiota of individuals collected in the same sampling site or different site are quite similar. The gut microbial community has low species richness and evenness, judging from the DGGE profiles. We found high percentage of cellulolytic organisms in bacterial culture from gut homogenate. Bacillus cereus, B. megaterium, B. thuringiensis, Microbacterium aurum, Pseudomonas plecoglossicida, and Aeromonas hydrophila were isolated from direct plating of gut homogenate. Stenotrophomonas maltophilia and M. binotii were obtained after filter paper enrichment procedure. These cellulolytic gut microbes may help the snail in degrading plant-derived cellulose-rich food. During 5 field surveys in Taroko National Park, highest occurrence of L. nitidum taivanum was seen on Arenga engleri (23%), but they are even more frequently found on off-tree non-living materials (46%). From dissecting observation, low diversity of gut microbiota, and high occurrence on off-tree locations, we hypothesize that L. nitidum taivanum is mainly saprophagous.
Microbial diversity in soil and bryophytes at Chi-Lan Mountain yellow cypress forest
Yu-Ning Wang (王郁甯)1, Chun-Yao Chen (陳俊堯)2, Shih-Chieh Chang (張世杰)1
Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien1
Department of Life Science, Tzu-Chi University, Hualien2
Tropical montane cloud forest (TMCF) is a unique ecosystem characterized by frequent fog. We selected Chi-Lan Mountain yellow cypress forest as a model to investigate the microbial diversity in TMCF ecosystem. In our 10 sampling sites along a transect, the average pH of the soil is 3.34±0.07 and average soil water content is 79.39±1.84%. Soil bacterial compositions are similar, but the archaeal and fungal communities are different. The dominant bacterial community in soil were Acidobacteria (35%) and Proteobacteria (27%), and archaeal community was dominated by Euryarchaeota (19%) and Crearchaeota (81%). Major soil cations and anions did not influenced composition of microbiota. The study area was covered with several bryophyte species. Bacterial and archaeal communities on bryophytes were host specific (ANOSIM, p<0.05), but there were no significant differences among plant locations (ANOSIM, p>0.05). Methane metabolic genes (mcrA and pmoA ) were detected in both soil and bryophytes. Nitrogen fixation (nifH), bacterial and archaeal ammonia oxidation (amoA) and nitrification (nirS, nirK, nosZ) genes were detected also in both soil and bryophytes, indicating that bryophyte-associated bacteria may be involved in nutrient cycling like soil bacteria. We conclude that bryophyte microbiota is likely ecologically important and should be taken into consideration when studying cloud forest ecosystem.
Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien1
Department of Life Science, Tzu-Chi University, Hualien2
Tropical montane cloud forest (TMCF) is a unique ecosystem characterized by frequent fog. We selected Chi-Lan Mountain yellow cypress forest as a model to investigate the microbial diversity in TMCF ecosystem. In our 10 sampling sites along a transect, the average pH of the soil is 3.34±0.07 and average soil water content is 79.39±1.84%. Soil bacterial compositions are similar, but the archaeal and fungal communities are different. The dominant bacterial community in soil were Acidobacteria (35%) and Proteobacteria (27%), and archaeal community was dominated by Euryarchaeota (19%) and Crearchaeota (81%). Major soil cations and anions did not influenced composition of microbiota. The study area was covered with several bryophyte species. Bacterial and archaeal communities on bryophytes were host specific (ANOSIM, p<0.05), but there were no significant differences among plant locations (ANOSIM, p>0.05). Methane metabolic genes (mcrA and pmoA ) were detected in both soil and bryophytes. Nitrogen fixation (nifH), bacterial and archaeal ammonia oxidation (amoA) and nitrification (nirS, nirK, nosZ) genes were detected also in both soil and bryophytes, indicating that bryophyte-associated bacteria may be involved in nutrient cycling like soil bacteria. We conclude that bryophyte microbiota is likely ecologically important and should be taken into consideration when studying cloud forest ecosystem.
Microbiota associated with rhodophyte Galaxaura filamentosa in Hualien
Tzu-Yin Chen (陳姿引), Chun-Yao Chen (陳俊堯)
Department of Life Science, Tzu-Chi University, Hualien
Epiphytic microbial communities associate closely with marine macroalga and invertebrates, and may have major impact on host health and fitness. Galaxaura filamentosa is a locally dominant rhodophyte in intertidal zone in Hualien, and its abundance does not have seasonal variation, therefore is a good model for studying rhodophyte microbiota. In this study we analyzed the epiphytic bacterial diversity on G. filamentosa. Samples taken from the top, middle, and lower parts of the same plant yielded highly similar microbial composition. Neighboring G. filamentosa individuals, individuals collected at different locations within the same pothole, and individuals collected from 2 adjacent potholes all showed similar microbial composition (ANOSIM, P>0.05), indicating diversification does not occur at meter-level spatial scale. Contrary to the microbial consistency in spatially different samples, individuals collected at different time showed temporal variation. We observed dramatic change in community composition between 2 samples collected in consecutive month. This indicates the composition of macroalgae microbiota is highly dynamic. Analysis of 16S rDNA clone library revealed that the dominant bacterial phyla on G. filamentosa were Cyanobacteria (71%), Proteobacteria (12%) and Bacteroidetes (4%). The dominant culturable bacteria genera as measured in summer of 2012 are Vibrio (80%) and Pseudoalteromonas. Our limited observation suggests that although this community is susceptible to environmental perturbation, it is highly resilient.
Department of Life Science, Tzu-Chi University, Hualien
Epiphytic microbial communities associate closely with marine macroalga and invertebrates, and may have major impact on host health and fitness. Galaxaura filamentosa is a locally dominant rhodophyte in intertidal zone in Hualien, and its abundance does not have seasonal variation, therefore is a good model for studying rhodophyte microbiota. In this study we analyzed the epiphytic bacterial diversity on G. filamentosa. Samples taken from the top, middle, and lower parts of the same plant yielded highly similar microbial composition. Neighboring G. filamentosa individuals, individuals collected at different locations within the same pothole, and individuals collected from 2 adjacent potholes all showed similar microbial composition (ANOSIM, P>0.05), indicating diversification does not occur at meter-level spatial scale. Contrary to the microbial consistency in spatially different samples, individuals collected at different time showed temporal variation. We observed dramatic change in community composition between 2 samples collected in consecutive month. This indicates the composition of macroalgae microbiota is highly dynamic. Analysis of 16S rDNA clone library revealed that the dominant bacterial phyla on G. filamentosa were Cyanobacteria (71%), Proteobacteria (12%) and Bacteroidetes (4%). The dominant culturable bacteria genera as measured in summer of 2012 are Vibrio (80%) and Pseudoalteromonas. Our limited observation suggests that although this community is susceptible to environmental perturbation, it is highly resilient.
Islands in an acid sea: bacteria living on plants growing on acidic grounds
Pei-Yu Hou (侯沛育)1, Yu-Han Kuo (郭瑀涵)1, Shih-Chieh Chang (張世杰)2, Po-Hsuan Chou (周帛暄)1, Chun-Yao Chen (陳俊堯)1
Department of Life Science, Tzu-Chi University, Hualien1
Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien2
Plant and soil represent two very different habitats, bacteria generally require different specialization in order to persist in either habitats. We hypothesize that bacteria capable of living in both soil and leaf are more prone to persist on plant leaves. We take the advantage of using plants growing on acidic soil to test this hypothesis, for soil bacteria need to survive in low pH condition but leaf-associated bacteria do not. We compared bacteria isolated from soil and vascular plant samples, collected from 3 organic farms (soil pH 4.6-4.8). Among the tested 402 leaf isolates, 78% of them grow on low pH media. Among 41 tested leaf isolates, 51% of them facilitated germination and growth of Arabidopsis, indicating that they have beneficial interaction with plants. The 5% indole-positive isolates are likely interacting with plant through phytohormone IAA production. We performed similar experiment using bacteria isolated from soil and bryophyte samples collected from yellow cypress cloud forest at Chi-Lan Mountain (soil pH 3.3). Among the tested 165 leaf isolates, only 7% of them could grow in low pH media. None of the soil or bryophyte isolates could produce indole. Our hypothesis is supported by data collected in drier acidic soils, but not that clear in humid acidic soil. We suspect that the high humidity and soil water content in cloud forest environment may help to overcome the dispersal barrier between plants.
Department of Life Science, Tzu-Chi University, Hualien1
Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien2
Plant and soil represent two very different habitats, bacteria generally require different specialization in order to persist in either habitats. We hypothesize that bacteria capable of living in both soil and leaf are more prone to persist on plant leaves. We take the advantage of using plants growing on acidic soil to test this hypothesis, for soil bacteria need to survive in low pH condition but leaf-associated bacteria do not. We compared bacteria isolated from soil and vascular plant samples, collected from 3 organic farms (soil pH 4.6-4.8). Among the tested 402 leaf isolates, 78% of them grow on low pH media. Among 41 tested leaf isolates, 51% of them facilitated germination and growth of Arabidopsis, indicating that they have beneficial interaction with plants. The 5% indole-positive isolates are likely interacting with plant through phytohormone IAA production. We performed similar experiment using bacteria isolated from soil and bryophyte samples collected from yellow cypress cloud forest at Chi-Lan Mountain (soil pH 3.3). Among the tested 165 leaf isolates, only 7% of them could grow in low pH media. None of the soil or bryophyte isolates could produce indole. Our hypothesis is supported by data collected in drier acidic soils, but not that clear in humid acidic soil. We suspect that the high humidity and soil water content in cloud forest environment may help to overcome the dispersal barrier between plants.
Microbial diversity associated with colonial Palythoa anemones in Hualien
Mei-Hsiu Wan (萬美秀), Yen-Chia Chen (陳彥嘉), Chun-Yao Chen (陳俊堯)
Department of Life Science, Tzu-Chi University, Hualien
Anthrozoans form the basis of coral reef ecosystem. Most studies focus on corals, however, anemones may have similarly important roles in the support and control of microbial communities and influence nutrient cycling of this ecosystem. We examined the anemones and their associated microbiota in Hualien in eastern Taiwan. Bacterial communities associated with colonial zoanthid anemones generally have higher species richness than solitary anemones. We found that clade C Symbiodinium is the dominant symbiotic dinoflagellates among several colonial anemones. We then selected Palythoa anemone, to reveal how spatial separation might affect the anemone-associated microbiota. Vibrios dominated the culturable bacterial community (93%). Various Vibrio species were isolated from the same individual. Pseudoalteromonas rubra was also isolated. Palythoa anemones usually colonize rock surface as compact clusters with sizes range from tens to hundreds of individuals. DGGE analysis based on 16S rDNA indicated that bacterial composition within a Palythoa colony is almost identical. DGGE profiles between nearby but separated colonies, or colonies located on different rocks, are also highly similar, indicating low beta-diversity in Palythoa-associated microbiota. The microbiota seems to be low in species evenness and dominated by one to a few bacterial species. We conclude that Palythoa-associated microbiota is low in alpha and beta diversity, and is usually dominated by vibrios.
Department of Life Science, Tzu-Chi University, Hualien
Anthrozoans form the basis of coral reef ecosystem. Most studies focus on corals, however, anemones may have similarly important roles in the support and control of microbial communities and influence nutrient cycling of this ecosystem. We examined the anemones and their associated microbiota in Hualien in eastern Taiwan. Bacterial communities associated with colonial zoanthid anemones generally have higher species richness than solitary anemones. We found that clade C Symbiodinium is the dominant symbiotic dinoflagellates among several colonial anemones. We then selected Palythoa anemone, to reveal how spatial separation might affect the anemone-associated microbiota. Vibrios dominated the culturable bacterial community (93%). Various Vibrio species were isolated from the same individual. Pseudoalteromonas rubra was also isolated. Palythoa anemones usually colonize rock surface as compact clusters with sizes range from tens to hundreds of individuals. DGGE analysis based on 16S rDNA indicated that bacterial composition within a Palythoa colony is almost identical. DGGE profiles between nearby but separated colonies, or colonies located on different rocks, are also highly similar, indicating low beta-diversity in Palythoa-associated microbiota. The microbiota seems to be low in species evenness and dominated by one to a few bacterial species. We conclude that Palythoa-associated microbiota is low in alpha and beta diversity, and is usually dominated by vibrios.
Environment is a major factor determining the composition of Polypedates braueri tadpole gut microbiota
Yung-Hsin Chang (張永欣), Chun-Yao Chen (陳俊堯)
Department of Life Science, Tzu Chi University, Hualien
Animals acquire their gut microbes from either the environment (environmental microbes and diet) or their parents. After entering the gut these bacteria are further selected according to host genetic background. Amphibian tadpoles serve an interesting model for gut ecosystem in that parent frogs have little chance to pass on their gut microbiota to their offsprings, therefore the microbiota might be more subjected to environmental influence. In this study we examine the gut microbiota of Polypedates braueri tadpoles cultured in microcosms with water collected from different ponds. Using 16S rDNA-based PCR/DGGE analysis, we found that all tadpole gut microbiota were different from that of eggs or water, indicating gut is a unique habitat. Tadpoles from the same family and kept in the same environment have almost identical DGGE profiles, but they developed unique microbiota when kept in different environments. When reared in the same environment, tadpoles from different families had gut microbiota similar in component species varied only in relative abundance. The dominant sequences in eggs were affiliated with Pseudomonas and Enterobacter, but changed to Akkermansia and Aeromonas in 1-month old tadpole gut, then switched back to Pseudomonas in 2-month old tadpoles. The results demonstrate high bacterial diversity in tadpole guts, and suggest that the composition of gut microbiota is affected by the environments, especially during early development.
Diversity in gut microbiota of Kurixalus eiffingeri tadpoles
Ya-Ting Hsu (許雅婷)1, Yung-Hsin Chang (張永欣)1, Chun-Yao Chen (陳俊堯)1, Yu-Chang Yang (楊育昌)2, Han-Chen Ho (何翰蓁)3
Department of Life Science1, Department of Anatomy3, Tzu-Chi University, Hualien
Endemic Species Research Institute, Council of Agriculture, R.O.C., Nantou2
Oviposition of eggs on arboreal microhabitats is a highly specialized form of anuran reproduction. Kurixalus eiffingeri utilize cut bamboo as their oviposition sites, and the hatched tadpoles can use the bamboo cup as a temporary pond. In this study we examined the gut microbiota of K. eiffingeri tadpoles collected from bamboo stomps and tree holes in northern and eastern Taiwan. Using 16S rDNA-based PCR-DGGE analysis, we discovered that tadpoles collected from the same bamboo stomp have highly similar gut microbiota. Gut samples share half of their DGGE bands with water collected inside the bamboo cup where the tadpoles lived, suggesting the connectivity between gut and water microbial communities. However, bacterial diversity in the water is much higher than that in tadpole guts. We analyzed two 16S rDNA libraries constructed with tadpoles collected from two bamboo stomps in the same area (B1 and B2 libraries), and another library of tadpoles kept in laboratory condition for several weeks (L library). Firmicutes, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes and Verrucomcicrobia are the dominant groups. However the relative abundance differs greatly between B1 and B2 libraries; B1 is dominated by Verrucomicrobia (79%), while B2 is dominated by Gammaproteobacteria (37%) and Betaproteobacteria (29%). The L library has high percentage of Gammaproteobacteria (60%). Aeromonas seems to be a common resident in tadpole gut. The universal appearance of novel Akkermancia-related sequences in most tadpoles is interesting, for these bacteria are potentially the mucin-degrader in K. eiffingeri tadpole gut community.
Department of Life Science1, Department of Anatomy3, Tzu-Chi University, Hualien
Endemic Species Research Institute, Council of Agriculture, R.O.C., Nantou2
Oviposition of eggs on arboreal microhabitats is a highly specialized form of anuran reproduction. Kurixalus eiffingeri utilize cut bamboo as their oviposition sites, and the hatched tadpoles can use the bamboo cup as a temporary pond. In this study we examined the gut microbiota of K. eiffingeri tadpoles collected from bamboo stomps and tree holes in northern and eastern Taiwan. Using 16S rDNA-based PCR-DGGE analysis, we discovered that tadpoles collected from the same bamboo stomp have highly similar gut microbiota. Gut samples share half of their DGGE bands with water collected inside the bamboo cup where the tadpoles lived, suggesting the connectivity between gut and water microbial communities. However, bacterial diversity in the water is much higher than that in tadpole guts. We analyzed two 16S rDNA libraries constructed with tadpoles collected from two bamboo stomps in the same area (B1 and B2 libraries), and another library of tadpoles kept in laboratory condition for several weeks (L library). Firmicutes, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes and Verrucomcicrobia are the dominant groups. However the relative abundance differs greatly between B1 and B2 libraries; B1 is dominated by Verrucomicrobia (79%), while B2 is dominated by Gammaproteobacteria (37%) and Betaproteobacteria (29%). The L library has high percentage of Gammaproteobacteria (60%). Aeromonas seems to be a common resident in tadpole gut. The universal appearance of novel Akkermancia-related sequences in most tadpoles is interesting, for these bacteria are potentially the mucin-degrader in K. eiffingeri tadpole gut community.
Will the Vibrio vulnificus mutants finally take over the sea?
Hwajiun Chen (陳華鈞)1, Wei-Chi Chen(陳維琪)2, Chun-Yao Chen (陳俊堯)2
Institute of Medical Sciences1, Department of Life Science2, Tzu-Chi University, Hualien
Vibrio vulnificus undergo phenotypic and genetic diversification when they are trapped in starvation. Certain phenotypes appear repeatedly in our starvation experiments. For strain 93U204, mutants with reduced motility appeared on day 8, and mutants with reduced proteolytic or hemolytic activities appeared on day 16. RpoS mutants were detected even later during starvation. Starved population of clinical isolate YJ016 showed similar sequence of phenotypic change, but at a faster pace. This indicates that the phenotypic succession is common in this species. We then tested whether V. vulnificus have similar phenotypic succession when they were starved in natural sea water (NSW). We adjusted NSW to salinity of 1%, 2% and 3%, and performed starvation experiments. 93U204 has the highest survival at 1% salinity NSW but for YJ016 the optimum is at 2%. Similar phenotypic succession was observed in both strains under all salinities. Competition experiments showed that these mutants outcompeted wildtype strain and have higher fitness under starvation. So why is the wildtype strain not outcompeted in their natural environment? We examined growth of rpoS mutant and wildtype strains of both 93U204 and YJ016, in high and low nutrient media. Although rpoS mutants grew faster in low nutrient media, they grew slower than wildtype strains. We examined 46 V. vulnificus marine isolates for motility, proteolytic and hemolytic activities. More than half of these bacteria have low motility, proteolytic or hemolytic activities, similar to our starvation mutants. This result suggests that phenotypic diversity is common in natural V. vulnificus population. We hypothesize that fluctuation in nutrient content may be an important force to maintain diversity in natural bacterial population.
Institute of Medical Sciences1, Department of Life Science2, Tzu-Chi University, Hualien
Vibrio vulnificus undergo phenotypic and genetic diversification when they are trapped in starvation. Certain phenotypes appear repeatedly in our starvation experiments. For strain 93U204, mutants with reduced motility appeared on day 8, and mutants with reduced proteolytic or hemolytic activities appeared on day 16. RpoS mutants were detected even later during starvation. Starved population of clinical isolate YJ016 showed similar sequence of phenotypic change, but at a faster pace. This indicates that the phenotypic succession is common in this species. We then tested whether V. vulnificus have similar phenotypic succession when they were starved in natural sea water (NSW). We adjusted NSW to salinity of 1%, 2% and 3%, and performed starvation experiments. 93U204 has the highest survival at 1% salinity NSW but for YJ016 the optimum is at 2%. Similar phenotypic succession was observed in both strains under all salinities. Competition experiments showed that these mutants outcompeted wildtype strain and have higher fitness under starvation. So why is the wildtype strain not outcompeted in their natural environment? We examined growth of rpoS mutant and wildtype strains of both 93U204 and YJ016, in high and low nutrient media. Although rpoS mutants grew faster in low nutrient media, they grew slower than wildtype strains. We examined 46 V. vulnificus marine isolates for motility, proteolytic and hemolytic activities. More than half of these bacteria have low motility, proteolytic or hemolytic activities, similar to our starvation mutants. This result suggests that phenotypic diversity is common in natural V. vulnificus population. We hypothesize that fluctuation in nutrient content may be an important force to maintain diversity in natural bacterial population.