Warming effects on dynamics of microbial communities in coastal waters of temperate and subtropical zones through dilution and fractionation experiments

THESIS Warming effects on dynamics of microbial communities in coastal waters of temperate and subtropical zones through dilution and fractionation experiments Dao Thi Anh Tuyet Graduate School of Science and Technology, Educational Division Department of Environment and Energy Systems Shizuoka University December 2014 THESIS Warming effects on dynamics of microbial communities in coastal waters of temperate and subtropical zones through dilution and fractionation experiments 海水温上昇が温帯および亜熱帯の沿岸海洋微生物群集に与える影響 〜特にガンマプロテオバクテリアに注目して Dao Thi Anh Tuyet 静岡大学 大学院自然科学系教育部 環境・エネルギーシステム専攻 2014 年 12 月 CONTENTS Chapter General introduction Introduction Methodological approaches References for chapter Chapter Effects of warming on microbial communities in coastal waters of temperate and subtropical zones in Northern Hemisphere with a focus on Gammaproteobacteria 10 2.1 Introduction 10 2.2 Materials and methods 13 2.2.1 Study sites and sample collection 13 2.2.2 Experimental processing 14 2.2.3 Total direct counts (TDCs) of prokaryotic abundance 15 2.2.4 Fluorescence in situ hybridization (FISH) 15 2.2.5 Growth rates 16 2.2.6 Enumeration of protists 17 2.2.7 Analysis of bacterial phylotypic community composition 17 2.3 Results 20 2.3.1 Environmental parameters 20 2.3.2 Warming effect on prokaryotes 21 2.3.3 Warming effect on bottom-up or top-down control by grazing 22 2.3.4 Warming effects on Gammaproteobacteria 24 2.4 Discussions 26 Acknowledgements 30 References for chapter 31 i Figures chapter 37 Table chapter 43 Supplementary chapter 46 Chapter Warming effect on the constituents of microbial communities 49 3.1 Introduction 49 3.2 Materials and methods 51 3.2.1 Sampling collection and experimental processing 51 3.2.2 Analysis of bacterial phylogenetic composition 51 3.2.3 Nucleotide sequence accession numbers 53 3.3 Results and discussion 54 3.3.1 Constituents of the bacterial communities in Suruga Bay 54 3.3.2 Constituents of the bacterial community in Ha Long Bay 56 References for chapter 58 Figure chapter 60 Conclusions and further works 69 Conclusions 69 Further works 70 Acknowledgements 71 ii Chapter General introduction Introduction Climate change is recognized as one of the most serious environmental concern for natural ecosystems and human activity As a consequence of climate change, sea temperature rose by 0.09 to 0.13 °C per decade over the period from 1971 to 2010 (IPCC 2013) The consequence will be more serious as climatic models predict that sea temperature will continue to increase with 24.8 °C during the 21st century (IPCC 2001, 2013) This warming may affect different aspects of the function and structure of marine ecosystems (Edwards and Richardson 2004, Montoya and Raffaelli 2010) particularly microbial food web Among the marine environment, prokaryotes and viruses present as the smallest constituents of ecosystem and to be considered to respond swiftly to the environmental change such as temperature Preceding studies indicated a positive correlation between temperature and bacterial abundance (Write and Coffin 1983, Lomas et al 2002) or growth rate (White et al.1991, Shiah & Ducklow 1994) However, controling mechanism of bacterial community through warming as bottom-up or top-down control by grazer and viral infection in a given community was not elucidated clearly Sjöstedt et al (2012) showed that a bacterial community shifted its constituents responding to the changes in temperature To show some aspects of microbial food web under the effect of warming, Sarmento et al (2010) summarized related studies and indicated that warming increases bacterial respiration and bacterial losses due to their grazers and bacterial production if nutrients are available These aspects of responses in microbial production may affect fisheries by changing the microbial loop and primary production which supply food resources for aquatic organisms such as fish, crustacean, mollusk and so on Climate change provides many extreme climatic events which may influence different aspects of ecosystem and environmental health Another necessity of microbial study related to environmental change stems from the fact that microbial community includes pathogens which may bring problems to aquatic organism and environmental health in coastal waters A pioneering study by Colwell (1996) clearly showed warming in surface temperature increased the incidence of cholera in Bangladesh A recent retrospective analysis suggested that sea surface temperature warming is favoring the spread of vibrios (Vezzulli et al 2012, 2013) which has different response to environmental factors than other heterotrophic bacterial population (Simidu et al 1987) Various studies have conducted until today on the warming effect on microbes, however, most study sites belonged to temperate (Shiah and Ducklow 1994, Apple et al 2006, Bouvy et al 2011, Sjöstedt et al 2012) or subarctic and Arctic regions (Lara et al 2013, Børsheim and Drinkwater 2014) Moreover, as the strongest ocean warming is predicted for the surface layer in tropical and subtropical regions of Northern Hemisphere (IPCC 2013), which might lead different effect on microbes in subtropical water compared to that in temperate zone Coastal area is regularly exposed to many anthropogenic influences such as fishery or aquaculture activities, transportation, tourism, harbor and nutrients supply Two studied sites were selected in this study belonging to temperate (Suruga Bay, Japan) and subtropical (Ha Long Bay, Viet Nam) coastal zones These two sites are completely different in climatic condition which may lead to different responses of microbial communities against changing in temperature As microbial ecological studies had been conducted in Suruga Bay (Takenaka et al 2007; Hao et al 2010) which provide sufficient information on the environmental control of prokaryotes, present study aimed to reveal warming effect on prokaryotes in this environment, which strengthen the understanding of findings supported with previous data, and become a control study for subtropical microbial ecology under the warming environment Bottom-up and top-down controls on prokaryotes under warming effect were also considered in complex interactions of a given community (chapter 2) As a response of potential pathogens to warming is considered to be major concern, the present study focused on Gammaproteobacteria which include various pathogenic bacteria (Brown and Volker 2004) In addition, bacterial 16S rRNA gene was employed to evaluate how the constituents of microbial community change in response to sea temperature rise (chapter 3) as an important issue, which has not yet reported from previous study Methodological approaches Microbes in coastal ecosystem are controlled by the availability of substrate supply (bottom-up control) and the mortality caused by grazers and/ or viral infections (top-down control) In order to elucidate the effect of warming on microbial communities, dilution and size fractionation methods were very well utilized (Landry and Hassett 1982, Wright and Coffin 1984, Rassoulzadegan and Sheldon 1986, Šolić and Krstulović 1994, Yokohama et al 2005) Top-down control is regularly determined by two methods: 1) comparing the values obtained with labeled bacteria as food tracers for natural assemblages as described in Sherr et al (1986), Wikner and Hagstrom (1988); 2) using size fraction method to estimate the difference in bacterial number/ growth between ungrazed (1µm) as reported by Wright and Coffin (1984), Coffin and Sharp (1987), Kuuppo-Leinikki (1990), Šolić and Krstulović (1994) Though the fractionation method tends to give somewhat lower grazing impact than the estimation from the experiment with labeled bacteria (Kuuppo-Leinikki 1990, Šolić and Krstulović 1994), the results of top-down control showed a similar trend regardless of the method employed (Šolić and Krstulović 1994) In present study, top-down control (by grazers) was estimated from difference in bacterial number/ growth between ungrazed (1µm) samples as referred by Wright and Coffin (1984), Coffin and Sharp (1987), Šolić and Krstulović (1994) Similarly to top-down control, bottom-up control on prokaryote is estimated from the differences in abundance and/ or growth rate of prokaryote between control dilution samples Dilution means sufficient nutrients as nitrogen and phosphate involved in marine water are given to bacteria Small size fraction of sea water consists bacteria, their grazers and viruses Not only protozoa, but larger grazers such as copepods exist in the community However, the most important grazers of bacteria controlling bacterial abundance was defined as small heterotrophic nanoflagellates/ grazer with theirs own size less than µm (Solic and Krstilovic 1994) Thus, in this study, pre-filtrate through larger pore-size filters of µm has not been conducted Observations from previous studies conducted in coastal water of Suruga Bay and Ha Long Bay indicated that annual seawater temperature varied about 14.6-16.4oC (Takenaka et al 2007, Hao et al 2010) and 12oC (Faxneld et al 2011), respectively Otherwise, climatic models predict that sea temperature will continue to increase with - 4.8 °C during the 21st century (IPCC 2001, 2013) Consequently, warming by or 5°C has been conducted in different seasons to estimate the impact of warming on microbial community in present study this phylum was not found in subtropical seawater of Ha Long Bay Among the composition of microbial community, Alphaproteobacteria and Cyanobacteria were globally dominated in surface seawater (Zinger et al 2011, Gilbert et al 2012) In this study, we confirmed the similar characteristics as Alphaproteobacteria dominated in the microbial communities in both temperate and subtropical coastal waters of Suruga Bay and Ha Long Bay Preceding studies indicated that warming promoted the spread of vibrio in coastal water (Cowell 2006, Vezzulli et al 2012, 2013) However, both qPCR and 16S rRNA sequence analyses did not detect any vibrio in this study Other related pathogenic clones such as Escherichia coli, Legionella spp were retrieved instead of vibrio Based on the analysis of 306 clones retrieved from the three experiments conducted in August 2011, October 2012 in Suruga Bay and in April 2013 in Ha Long Bay, the constituents of microbial communities changed by warming in both temperate and subtropical coastal waters Gammaproteobacteria tended to predominate by warming which included some specific species as Escherichia coli, Legionella sp A possibility to increase potentially pathogenic prokaryotes by warming was suggested 57 References for chapter Altschul SF, TL Madden, AA Schäffer, J Zhang, Z Zhang, W Miller, JD Lipman (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 25:3389–3402 Colwell RR (1996) Global climate and infectious disease: the cholera paradigm Science 274:2025–2031 Gilbert JA, JA Steele, JG Caporaso, L Steinbrück, J Reeder, B Temperton, S Huse, AC McHardy, R Knight, I Joint, PJ Somerfield, JA Fuhrman, D Field (2012) Defining seasonal marine microbial community dynamics ISME J 6:298-308 Lane JD (1991) 16S/23S rRNA sequencing, p115–175 In: Stackebrandt E, Goofellow M, editors Nucleic acid techniques in bacterial systematics John Wiley & Sons, Chichester, United Kingdom Pinhassi J, A Hagström (2000) Seasonal succession in marine bacterioplankton Aquatic Microbial Ecology 21: 245-256 Saitou N, M Nei (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees Mol Biol Evol 4:406–425 Schloss PD, J Handelsman (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness Appl Environ Microbiol 71:1501–1506 Sjöstedt J, Å Hagström, UL Zweifel (2012) Variation in cell volume and community composition of bacteria in response to temperature Aquatic Microbial Ecology 66(3):237–246 Thompson JD, DG Higgins, TJ Gibson (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence 58 weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22:4673–4680 10 Vázquez-Domínguez E, D Vaqué, JM Gasol (2012) Temperature effects on the heterotrophic bacteria, heterotrophic nanoflagellates, and microbial top predators of the NW Mediterranean Aquat Microb Ecol 67:107-121 11 Vezzulli L, I Brettar, E Pezzati, PC Reid, RR Colwell, MG Hofle, C Pruzzo (2012) Long-term effects of ocean warming on the prokaryotic community: evidence from the Vibrios ISME J 6(1):21–30 12 Vezzulli L, RR Colwell, C Pruzzo (2013) Ocean warming and pread of pathogenic Vibrios in the aquatic environment Microb Ecol 65:817–825 13 Zinger L, Amaral-Zettler LA, Fuhrman JA, Horner-Devine MC, Huse SM, David B Welch M, Martiny JBH, Sogin M, Boetius A, Ramette A (2011) Global patterns of bacterial beta-diversity in seafloor and seawater ecosystems PLoS One 6(9):e24570 59 Figure chapter +5°C Dil (n=14) In situ temp Dil (n=13) All temp After incubation (48h) Initial (n=33) a Cont (n=24) 0% 20% 40% 60% 80% 100% Actinobacteria Planctomycetes Bacteroidetes Verrucomicrobia Alphaproteobacteria Gammaproteobacteria unclassified Bacteria unclassified root b 4.00 Suruga Bay-August 2011 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Cont (n=24) Dil (n=13) Dil (n=14) All temp In situ temp +5°C Initial (n=33) After incubation (48h) Fig (a) Phylogenetic analysis of bacterial communities and (b) diversity indices among bacterial communities of different treatments for the experiment conducted in Suruga Bay in August 2011 (n=xx): numbers of sequenced clones 60 Proteobacteria Actinobacteria Bacteroidetes Planctomycetes I 61 Verrucomlcrobla Fig Phylogenetic tree of planktonic Bacterial 16S rRNA gene sequences for the experiment conducted in August 2011 in Suruga Bay Red letter indicates candidate of pathogens of human and aquatic organisms OTU is expressed by sampling site (S: Suruga Bay), month of sampling (Au: August), incubation period (from to 48 h), and incubation conditions as control (C) and dilution (D) at in situ temperature (0), and at in situ plus 5°C (5) (X/XX) indicated contribution of clones per total clones Bootstrap indicates percent (counted 1,000) 62 +5°C Dil (n=28) Cont (n=29) In situ temp After incubation (24h) Initial (n=17) a Dil (n=23) Cont (n=23) 0% Cyanobacteria/Chloroplast Bacteroidetes unclassified Bacteria 20% 40% 60% TM7 Alphaproteobacteria 80% 100% Planctomycetes Gammaproteobacteria b Suruga Bay-October 2012 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Cont (n=23) Dil (n=23) In situ temp Initial (n=17) Cont (n=29) Dil (n=28) +5°C After incubation (24h) Fig (a) Phylogenetic analysis of bacterial communities and (b) diversity indices among bacterial communities of different treatments for the experiment conducted in Suruga Bay in October 2012 (n=xx): numbers of sequenced clones 63 10.02 1000 S.Oc-0-1 (1/17) S.Oc-2400-11 (4/23) S.Oc-0-2 (2/17) S.Oc-2400-3 (4/23) S.Oc-24C0-4 k5/23) !: S.Oc-2405- (11 28) "C ::r S.Oc-2405-4 (2/28) Ql Rhodobacteraceae bacterium (AB362415) "C , S.Oc-24C0-1 (2/23) ' (I) ~ ~okt9Jcella s"diminiq,torif ~1 1J~8) ose1 onoce a pacJJca A 60 0" Marivita litorea (EU512918) Ql S.Oc-24C0-131 )11 /23) S.Oc-24C5-27 (2 29) (I) , S.Oc-24C5-3 (2/29) iii' Phaeobacter gallaeciesis (AY 136134) Roseovarius nubinhibens (JQ905065) S.Oc-0-3 ~1 /17) S.Oc-0-21 (2/ 7) Alpha proteobacterium (FJ532493) S.Oc-0-22 (1 /17l Oceanospirillales bacterium HQ675262) Proteobacteria S.Oc-24C5-29 ~2/29) S.Oc-24C5-7 (312 ) Escherichia coli (JQ951605) S.Oc-2405-18 ~2/28) Cl S.Oc-24C5-32 (1 /29 Ql Alteromonas alvinellae (AY13611 3) •3 S.Oc-2400-1 (1 /23) Ql S.Oc-24C0-21 (1/23) "C , Haliea sp (AB646260) S.Oc-2400-12 (1/23) (I) Methylocaldum sze%ediensis (U89300j Methylococcaceae acterium (AB636 99) 0" Ql S.Oc-24C5-28 (1129) S.Oc-24C5-19 (1/29) (I) , S.Oc-24C5-14 (2129) iii' Solemya pervernicosa gill symboint (AB499617) Coxiella burnetii (089800) Le~ionella steelei (JX206466) S.Oc-0- (3/17) S.Oc-2405-17 (2/28) S.Oc-0-15 (1/17) Cloacibacterium rupense (EU581834) S.Oc-2400-15 (4123) Lishizhenia tianpnenensis (EU183317) S.Oc-2405-12 (1/28) S.Oc-24C5-16 (2/29{ S.Oc-2400-16 ~ 123) S.Oc-24C5-9 ~ / 9) Flavobacteria acterium (AB557547) S.Oc-24C5-2 (1 /29) S.Oc-24C5-31 ~1 /29) S.Oc-24C0-132 ( /23) S.Oc-0-14 (1117~ Aequorivita sg (F 691437) S.Oc-24 5:.S (1 /28) S.Oc-24C5-1 (1 /29) S.Oc-2400-18 ~2/23) S.Oc-2405-21 (2 28) S.Oc-2400-2 (5/23) Algibacter agarolyticus (JN864027) Bacteroidetes Psychroserpens_sp (JQ660965) Gaetbulibacter jejuensis ( FJ490367~ Meridianimaribacter flavus (FJ3606 4) Flavobacteriaceae bacterium (HQ882589) S.Oc-2405-7 (1/28) Bacteroidetes bacterium (JF488472) S.Oc-2405-15 ~/28) S.Oc-2405-24 (1 8) S.Oc-24C5-11 (8/29) S.Oc-24C0-3 t1 /23) S.Oc-0-1 (1/1 ) S.Oc-0-8 (1117) TM7 phylum sp (GU412737) S.Oc-0-18 (2117) Synechococcus sp (FJ497741) Cyanobacteria S.Oc-24C0-5 ~2/23) Rhodopire lular baltica (HQ845529) Planctomycetes S.Oc-0-23 ~1 / 17) S.Oc- 4C5-25 ~ /29) Aquifex pyrophilus (M83 48) ~ 1000 ~ ~ 1000 837 930 1000 757 1000 1000 1000 ITM7 I I 64 Fig Phylogenetic tree of planktonic Bacterial 16S rRNA gene sequences for the experiment conducted in October 2012 in Suruga Bay Red letter indicated candidate of pathogens of human and aquatic organisms OTU is expressed by sampling site (S: Suruga Bay), month of sampling (Oc: October), incubation period (from to 24 h), and incubation conditions as control (C) and dilution (D) at in situ temperature (0), and at in situ plus 5°C (5) (X/XX) indicated contribution of clones per total clones Bootstrap indicates percent (counted 1,000) 65 +5°C Dil (n=41) Cont (n=11) In situ temp After incubation (20h) Initial (n=20) a Dil (n=15) Cont (n=18) 0% Chloroflexi Cyanobacteria/Chloroplast Verrucomicrobia Gammaproteobacteria 20% 40% 60% Acidobacteria Planctomycetes Betaproteobacteria unclassified Bacteria 80% 100% Actinobacteria Bacteroidetes Alphaproteobacteria b Ha Long Bay-April 2013 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 Cont (n=18) Dil (n=15) In situ temp Initial (n=20) Cont (n=11) Dil (n=41) +5°C After incubation (20h) Fig (a) Phylogenetic analysis of bacterial communities and (b) diversity indices among bacterial communities (bottom panel) of different treatments for the experiment conducted in Ha Long Bay in April 2013 (n=xx): numbers of sequenced clones 66 Phaeobacter caeruleus (KC176242) H.Ap-0-25 (1120) H.Ap-0-6 (4/20) Citreicel/a marina (KC534395) H.Ap-2005-45 (3/41) H.Ap-2005-33 (5/41) H.Ap-2005-20 (2/41) Seohaeico/a saemankumensis (EU221274) Sulfitobacter dubius (KC534303) Roseovarius aestuarii (HQ441 227) H.Ap-0-5 (2/20) Celeribacter neptunius (FJ535354) H.Ap-2005-11 (2/41) Tateyamaria omphalii (AB193438) H.Ap-20C0-1 (1 /18) l> -a::r Ill , "C