Vol. 72: 215–225, 2014 AQUATIC MICROBIAL ECOLOGY Published online June 12 doi: 10.3354/ame01696 Aquat Microb Ecol
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Linkage between copepods and bacteria in the North Atlantic Ocean
Daniele De Corte1,*,**, Itziar Lekunberri1,**, Eva Sintes1, Juan Antonio L. Garcia1, Santiago Gonzales2, Gerhard J. Herndl1,2
1Department of Limnology and Oceanography, Center of Ecology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria 2Department of Biological Oceanography, Royal Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB, Den Burg, The Netherlands
ABSTRACT: Copepods and bacteria are fundamental components of the pelagic food web and play a major role in biogeochemical cycles. Marine bacteria have a free-living or particle- attached lifestyle, but as members of the microbial food web, the only biotic interaction of bac- teria is commonly assumed to be with their predators (protists and/or viruses). However, a copepod’s body is highly enriched in organic matter and harbors a large and complex bacterial community. The aim of this study was to compare the composition of the free-living bacterial community of the open Atlantic to that associated with copepods. We used 454 high- throughput sequencing of the 16S rRNA gene to decipher the bacterial community composition associated with this zooplankton group and the ambient water. Significant differences were found between the bacterial communities associated with the dominant copepod families (Calanoida: Centropagidae and Clausocalanidae; Cyclopoida: Corycaeidae, Oncaeidae, and Lubbockiidae) and the ambient water. Bacilli and Actinobacteria dominated the copepod- associated community and Alphaproteobacteria, Deltaproteobacteria, and Synechococcus dom- inated the free-living community. However, the presence of shared bacterial operational taxo- nomic units (OTUs) between these 2 distinct habitats suggests a dynamic exchange of bacteria between seawater and copepods. Taken together, our results support the hypothesis that the interior and exterior surfaces of copepods provide a specific niche with a strong selective pres- sure for bacteria.
KEY WORDS: Microbes · Zooplankton · Open ocean · 454 pyrosequencing
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INTRODUCTION bacterial abundances associated with various microhabitats such as particles, aggregates, fecal Prokaryotes constitute the largest fraction of liv- pellets, and zooplankton exceeding those of free- ing biomass in marine ecosystems and are the living bacteria (Simon et al. 2002, Tang et al. main force driving biogeochemical cycles (Azam et 2010). Also, attached bacteria commonly exhibit al. 1983). In the oceanic water column, the majority high growth rates and enzymatic activities (Karner of the prokaryotes are free-living, with commonly & Herndl 1992, Grossart et al. 2006). The micro- less than 5% of total prokaryotic cells associated habitats of these attached bacteria are frequently with aggregates (Bell & Albright 1982, Unanue et characterized by concentrations of organic matter al. 1992). However, several studies have reported and inorganic nutrients that are orders of magni-
*Corresponding author: [email protected] © Inter-Research 2014 · www.int-res.com **Both authors contributed equally to this work 216 Aquat Microb Ecol 72: 215–225, 2014
tude higher than in the surrounding water (Bo ch - MATERIALS AND METHODS dansky & Herndl 1992, Alldredge 2000, Grossart & Tang 2010, Tang et al. 2010). These distinctly dif- Sampling of ambient water ferent microhabitats may favor biogeochemical reactions that otherwise would not occur in the Water samples were collected during the MEDEA- oceanic water column (Grossart & Tang 2010). The I cruise (October 2011) on board RV ‘Pelagia’ at 4 sta- anoxic and hypoxic conditions found in some tions along a latitudinal transect in the North Atlantic pelagic aggregates, animal guts, and fecal pellets from 24° 40’ N, 34° 56’ W to 30° 27’ N, 24° 32’ W (See favor anaerobic reactions not occurring in the sur- Fig. S1 in the Supplement at www.int-res.com/ rounding oxygenated water (Alldredge & Cohen articles/suppl/a072p215_supp.pdf). 1987, Deangelis & Lee 1994, Grossart & Tang Seawater samples were collected with a Seabird 2010, Tang et al. 2011). Furthermore, several stud- conductivity-temperature-depth (CTD) rosette sam- ies have shown that the interactions between pler equipped with 18 Niskin bottles (25 l each). To prokaryotes and predators such as protists and determine the bacterial community composition of viruses are substantially different in these micro- the ambient seawater, 10 l of water were sampled habitats as compared to the ambient seawater from the lower euphotic zone (about 100 m depth) (Caron 1987, Riemann & Grossart 2008, Grossart & and from ~750 m depth. Tang 2010). The seawater was filtered through a 0.2 µm GTTP Marine copepods provide a complex microhabitat membrane filter (Millipore) and subsequently, the fil- in marine ecosystems, with their complex body struc- ters were stored at −80°C until further processing in ture and extensive surface potentially available for the laboratory. Although these samples include some microbial colonization (Tang et al. 2010). Further- particle-attached bacteria, the free-living community more, copepods contribute to the microbial food web is dominant (Bochdansky et al. 2010). Thus, we refer through the release of biologically available dis- to the ambient seawater bacterial community as the solved organic matter and nutrients during the diges- free-living community. tive processes (Azam et al. 1983, Møller et al. 2003, Tang 2005, Møller 2007, Tang et al. 2010). In contrast to detrital particles, copepods can collect organic Sampling of zooplankton compounds and cells through the ingestion of food, thereby allowing a continuous production and re - Zooplankton were collected at the same stations as lease of prokaryotes through their fecal pellets (Tang the ambient water using vertical plankton tows 2005). Moreover, many zooplankton species perform (200 µm mesh size, hoisted at 30 m min−1) from 750 m vertical migration (Kobari & Ikeda 2001). This migra- to the surface. Water samples were collected at the 2 tion in stratified waters favors the dispersal and depth layers (~100, ~750 m) within which the cope- acquisition of microbes from different water layers pods migrate during the diel cycles (Steinberg et al. and allows them to cross physical barriers, such as 2000, Tang et al. 2010), to compare the composition pycnoclines (Grossart et al. 2010). Several abundant of the free-living bacterial community with the zoo- open-ocean copepods exhibit diel vertical migration plankton-associated bacterial community obtained potentially favoring dispersal of copepod-associated from the integrated net tows. bacteria over the euphotic to mid-mesopelagic layers The content of the cod end of the plankton net was (Steinberg et al. 2000, Grossart et al. 2010, Tang et al. transferred into a plankton splitter and then concen- 2010). trated over a 70 µm mesh Nitex screen. The zoo- The aim of this study was to compare the phyloge- plankton samples were then transferred into 2 ml netic composition of the bacterial community associ- Eppendorf tubes and stored at −80°C until sorting. In ated with copepods collected in the North Atlantic the laboratory, the zooplankton were thawed to room Ocean to that of the ambient water using 454 pyrose- temperature, transferred to a Petri dish filled with quencing. Specifically, we hypothesized that a vari- 0.2 µm filtered seawater for sorting of the dominant able transient bacterial community is present in the copepod taxa (Calanoida: Centropagidae and Clau- copepods in addition to a stable resident community. socalanidae; Cyclopoida: Corycaeidae, Oncaeidae, The transient community reflects the composition of and Lubbockiidae). To evaluate the gut-associated the ambient water, while the resident community is bacteria of different copepods, 10 individuals of each specifically adapted to the microenvironmental con- taxon were collected and washed 3 times with Milli- ditions in the copepods. Q water to remove bacterial cells associated with the De Corte et al.: Links between copepods and bacteria 217
external surface of the copepods. Subsequently, the cedure pipeline of Mothur software, version 1.31 copepods were transferred into sterile Eppendorf (Schloss 2009). The 16S rDNA pyrotags were sorted tubes for nucleic acid extraction. according to their respective barcode into the differ- ent samples. The raw sequence reads were filtered, trimmed, and quality checked, and sequences DNA extraction smaller than 200 bp were discarded. Subsequently, the sequences were aligned with the SILVA data- The DNA of the ambient-water samples was base, and the pairwise distance matrix was calcu- extracted using an Ultraclean Soil DNA isolation Kit lated. The 16S rDNA sequences with a 97% se - (MoBIO Laboratories). The DNA from the copepod quence similarity were clustered into operational samples was extracted using a phenol-chloroform taxonomic units (OTUs). Taxonomic assignment was extraction protocol (Weinbauer et al. 2002), preceded performed using QIIME (Caporaso et al. 2010), and by a bead-beating step to facilitate lysis of the cope- all unclassified bacteria at the phylum level were dis- pods. To check the quality of the DNA following carded. Additionally, MEGAN (Huson et al. 2007) extraction, a NanoDrop ND-1000 spectrophotometer was used to build the hierarchical phylogenetic tree (NanoDrop Technologies) was used. of the bacterial community as an alternative to QIIME for taxonomic identification. The MEGAN analysis was based on BLAST results (Altschul et Pyrosequencing of the 16S rDNA al. 1997) against SILVA and Greengenes databases bacterial community (data not shown) following the NCBI taxonomy (Say- ers et al. 2012). Rarefaction curves, Chao1, ACE rich- The 16S rRNA genes (16S rDNA) of the zooplankton ness, and the Shannon index of diversity were calcu- and ambient-water samples were PCR amplified with lated with Mothur (Schloss 2009). the bacterial primers 341f (5’-CCT ACG GGA GGC Pairwise UniFrac distance and principal coordinate AGC AG-3’) and 907r (5’-CCG TCA ATT CMT TTG analysis (PCoA) (Lozupone & Knight 2005) were used AGT TT-3’) (Muyzer et al. 1998, Grossart et al. 2009). to compare the bacterial community composition in The PCR amplification of the 16S rRNA gene of the the different samples (implemented in QIIME). The samples was carried out in a 50 µl reaction volume us- established phylogenetic tree was built with Mothur ing Fermentas Taq polymerase (Thermo Scientific) in (Schloss 2009), and the Unifrac distance matrix was a Mastercycler (Eppendorf) with the following param- calculated with FastUnifrac (Lozupone & Knight eters: initial denaturation at 94°C for 3 min, followed 2005). The Unifrac distance matrix was calculated by 30 cycles of denaturation at 94°C for 1 min, an- unweighted using only presence−absence informa- nealing at 55°C for 1 min, and extension at 72°C for tion of bacterial OTUs, or weighted and thus taking 1 min, with a final extension at 72°C for 7 min. The the relative proportion of each bacterial OTU to the PCR products were additionally purified with a PCR total bacterial community into account. A t-test (im- purification kit (5-Prime). The quality of the PCR plemented in Sigma Plot v.11) was used to test for the product was checked on 2% agarose gel. The 16S statistical difference between samples. rDNA amplicons were subsequently sequenced in a Roche 454 GS Junior next generation sequencing platform based on the Titanium chemistry by IMGM RESULTS Laboratories GmbH (Martinsried, Germany). All sam- ples were barcoded using multiplex identifiers and Analysis of the pyrosequencing library sequenced together in 1 run. The resulting sequences were divided into 4 groups: 2 orders of copepods The 454 pyrosequencing analysis was performed to (Calanoida and Cyclopoida) and 2 water layers (Deep, investigate the differences between the bacterial corresponding to ~750 m; and Surface, corresponding community composition associated with 2 orders of to 100 m; see Table S1 in the Supplement). copepods (Cyclopoida and Calanoida) and the bacte- rioplankton community collected from the 2 bound- ary depth layers (~750 and 100 m depths) at the same Bioinformatic analysis and phylogenetic classification location as the copepods were collected. In total, we obtained 65 855 reads for the entire set of samples The bioinformatic analysis of the 16S rDNA se - with an average length of 450 bp. The trimming of quences largely followed the standard operating pro- low-quality reads resulted in 25 101 sequences with 218 Aquat Microb Ecol 72: 215–225, 2014
an average length of 307 bp used for Table 1. Total number of operational taxonomic units (OTUs; cutoff 97% simi- further analyses (Table S1 in the Sup- larity), Chao and Ace species richness, and Shannon and Simpson diversity indices obtained from 16S rDNA sequence libraries from ambient-water and plement). From the total number of copepod-associated bacteria trimmed sequences, 3970 and 12 978 reads were categorized as unique at Sample OTUs Chao Ace Shannon Simpson the 97% and 100% similarity level, observed (1-D) respectively. Calanoida St.1 196 258 276 4.18 0.97 St.1 151 231 215 3.88 0.95 OTU richness in ambient water Cyclopoida and copepods St.2 162 276 275 4.18 0.97 St.2 153 206 206 4.12 0.97 The rarefaction analysis showed St.3 132 181 170 4.14 0.97 St.3 189 306 346 4.36 0.97 different trends for the 2 sets of sam- St.4 193 279 341 4.40 0.98 ples (Fig. 1). While the rarefaction St.4 65 120 210 3.25 0.91 curves for the copepod samples St.4 116 226 386 3.86 0.95 approached a plateau, the rarefaction Water samples Stn 1_750 m 486 2268 3707 5.33 0.98 curves of the ambient water samples Stn 1_100 m 358 1740 3755 4.65 0.96 did not level off (Fig. 1). Hence, the Stn 2_900 m 470 3026 7600 5.30 0.98 sequencing effort was sufficient to Stn 2_100 m 292 1507 3115 4.30 0.94 sample most members of the bacterial Stn 3_100 m 258 791 1303 4.15 0.94 Stn 4_750 m 493 2081 4830 5.28 0.98 community associated with the cope- Stn 4_100 m 346 1683 2716 4.50 0.95 pods, while it was not sufficient for the ambient water samples. More- over, these results indicate a lower diversity in the were computed for each sample using the 97% simi- copepod-associated bacterial community as com- larity threshold. These indices also indicated a higher pared to the ambient-water community (Table 1). diversity in the bacterial community of the ambient The Chao richness index estimated, on average, water as compared to the copepod-associated com- 231 ± 57 (average ± SD) OTUs (ranging from 120− munity (Table 1). Two of the 3 samples of the Cyclo- 306, N = 9) for the copepod-associated bacterial com- poida-associated bacterial community collected at munities and 1870 ± 693 OTUs (ranging from 791− Stn 4 exhibited significantly lower (t-test, p < 0.01) 3026, N = 7) for the ambient water (Table 1). Similar diversity than the other copepod samples. These 2 results were obtained with the ACE richness index samples of Cyclopoida had the lowest number of (Table 1). Shannon and Simpson diversity indices OTUs (t-test, p < 0.01) and the lowest Shannon (t-test, p = 0.01) and Simpson indices (t-test, p < 0.01) of the 400 entire dataset (Table 1). In the ambient water, the bacterial community at 100 m depth exhibited a lower diversity (t-test, p = 300 0.02), a lower number of OTUs (t-test, p = 0.02) and lower Shannon (t-test, p < 0.01) and Chao (t-test, p = 0.03) indices than that of the 750 m layer, although 200 the ACE index was not significantly different be - tween these depth layers (t-test, p = 0.86; Table 1). The most abundant bacterial OTU of the ambient 100 water contributed on average 25% to the total num- Copepod-associated ber of ambient-water OTUs, while the most abun-
Number of estimated OTUs Ambient water dant copepod-associated bacterial OTU contributed 0 0 200 400 600 800 1000 1200 1400 1600 only 18% to the total copepod-associated bacterial Number of 16S rDNA sequences OTUs (see Fig. S2 in the Supplement). Singletons (OTUs appearing only once in the entire pyrose- Fig. 1. Rarefaction curves obtained for the 16S rDNA se- quences of calanoid and cyclopoid copepod-associated and quencing library) accounted for 27% of the total ambient-water bacterial communities. Operational taxo- number of bacterial OTUs of the ambient water, but nomic units (OTUs) were defined at 97% sequence identity only 8% of the copepod-associated OTUs (Fig. S2). De Corte et al.: Links between copepods and bacteria 219
The bacterial communities of the ambient water were dominated by a few very abundant OTUs and a Pseudomonadales large number of rare OTUs (Fig. S2). In contrast, members of the copepod-associated bacterial com-
munity were more evenly distributed than those of ± 0.1 1.0 ± 0.8 Oceanospirillales the ambient water with a comparatively low number of rare OTUs (Fig. S2).
Chromatiales Composition of the bacterial community in the ambient water and in copepods Synechococcales The phylogenetic analysis of the 25 101 sequences performed in QIIME using the Greengenes database revealed a clear clustering into 4 groups of bacterial Chloroplasts
communities (ambient water at 100 m and 750 m and Cyanobacteria Gammaproteobacteria calanoid and cyclopoid copepods; Table 2). Firmi- cutes contributed 23% and 27%, Ac tino bacteria 22% and 19%, and Alphaproteobacteria 20% and 11% to Clostridia the Calanoida- and Cyclopoida-associated bacteria, respectively. Betaproteobacteria contributed 1.5% and 16% to the Calanoida- and Cyclopoida-associ- Lactobacillales ated bacteria, respectively (Fig. 2a). This relatively Firmicutes high abundance of the Betaproteobacteria in cyclo - poid copepods was mainly caused by 2 samples col- Bacilli lected at Stn 4 where Betaproteobacteria contributed 53% to Cyclopoida-associated bacteria (Fig. 2a). Generally, the copepod-associated community was Koll 13 characterized by a relatively high contribution of chloroplasts, probably derived from ingested phyto- plankton (7 ± 8%; Fig. 2a). Although the composition of the bacterial commu- Acidimicrobiales nity of the ambient water was rather uniform among the different stations, as was the composition of Actinobacteria the copepod-associated bacterial community, 2 sam- Actinomycetes ples of Cyclopodia-associated bacterial communities were strikingly different from all other samples (Fig. 2a), specifically the bacteria associated with the Rickettsiales families Oncaeidae and Lubbockiidae at Stn 4, although members of the Oncaeidae were also collected at other stations. These 2 samples were composed mainly of Betaproteobacteria (genus Burk- Caulobacterales holderiales, 51%) and Flavobacteria (genus Flavo-
bacteriales, 16%; Fig. 2a,b). layers differentdepth 2 from collected water ambient of samples from and orders copepod 2 with associated The bacterioplankton community of the 100 m Rhodobacterales depth layer mainly consisted of Alphaproteobacteria
(30%), Cyanobacteria (32%; mainly composed of Alphaproteobacteria Synechococcales , 31%), and Actinobacteria (17%). Rhizobiales The bacterial community of the 750 m layer was mainly composed of members of Deltaproteobac - teria (29%), Alphaproteobacteria (20%), Chloroflexi (10%), SAR406 (10%), and Gammaproteobacteria (6%; Fig. 2a, Table 2). Cyclopoida 3.2 ± 3.0 3.6 ± 2.6Surface 3.4 ± 2.6Deep 0.7 ± 0.5 1.2 ± 0.6 19.1 ± 11.4 2.0 ± 0.6 0.1 ± 0.1 ± 0.1 ± 8.6 ± 5.2 14.9 ± 9.4 0.4 ± 0.2 3.5 ± 4.1 – 4.4 ± 4.9 26.9 ± 4.5 0.2 ± – 0.1 ± 0.5 0.7 ± 0.2 ± 1.6 ± 0.9 18.8 ± 5.2 2.2 ± 3.9 15.0 ± 6.9 – – 4.0 ± 1.6 0.1 ± – – – – 0.3 ± 0.1 31.4 ± 11.3 0.2 ± 0.3 0.2 ± 0.1 – – – 0.1 ± 0.2 4.0 ± 1.9 1.5 ± 0.9 – Calanoida 5.3 ± 2.7 11.4 ± 12.1 1.5 ± 0.1 1.5 ± 1.8 21.7 ± 1.8 – 0.3 ± 0.4 8.1 ± 1.8 8.5 ± 2.1 6.7 ± 3.9 16.1 ± 15.3 0.3 ± 0.2 0.6 ± 0.4 0.2 Table Table 2. Phylogenetic affiliation and relative contribution (%, ± SD) of the individual bacterial families to the total number of sequences obtained from the communities 220 Aquat Microb Ecol 72: 215–225, 2014
a 100
80 Alpha Beta Gamma Delta 60 Flavobacteria Actinobacteria Clostridia 40 Bacilli SAR406 Synechococcophycideae
Chloroplast % of 16S rDNA sequences 20 Cloroflexi Others
0 St.1 St.1 St.2 St.2 St.3 St.3 St.4 St.4 St.4 St.1_780 m St.1_100 m St.2_900 m St.2_100 m St.3_100 m St.4_750 m St.4_100 m
Oncaeidae Oncaeidae Oncaeidae Water samples Corycaeidae Corycaeidae Corycaeidae Lubbockiidae Centropagidae Clausocalanidae Calanoida Cyclopoida b 100
80 Rickettsiales Rhodobacterales Burkholderiales Chromatiales 60 Delta_Others Desulfobacterales Flavobacteriales 40 Actinomycetales Clostridiales Bacillales % of 16S rDNA sequences Streptophyta 20 Synechococcales Lactobacillales Others 0 St.1 St.1 St.2 St.2 St.3 St.3 St.4 St.4 St.4 St.1_780 m St.1_100 m St.2_900 m St.2_100 m St.3_100 m St.4_750 m St.4_100 m Water samples Oncaeidae Oncaeidae Oncaeidae Corycaeidae Corycaeidae Corycaeidae Lubbockiidae Centropagidae Clausocalanidae Calanoida Cyclopoida
Fig. 2. Relative contribution of the more abundant phylogenetic (a) classes and (b) orders to the total number of 16S rDNA sequences obtained from copepod-associated and ambient-water bacterial communities De Corte et al.: Links between copepods and bacteria 221
Acidimicrobiales SURFACE Actinobacteria Actinomycetales DEEP environmental samples
To investigate an alternative to the QIIME classifier PCoA – PC1 vs PC2 and to directly visualize the distribution of individual bacterial taxa among the 2 copepod orders and the 2 depth layers, the samples were blasted against the SILVA and Greengenes database (data not shown). These results were visualized by MEGAN (Huson at al. 2007) using the BLAST hit-score to assign the tax- onomy. As indicated in Fig. 3, some bacterial taxa associated with the 2 copepod orders were not pres- ent in the water column, including members of Firmi- cutes, Fusobacteriales, and most of the Betaproteo- bacteria. At the genus level, bacteria associated with copepods but absent in the ambient water belonged to the Actinomycetales, Bifidobacteriales, Bacte roi- Cyclopoida dales, Deinicoccus, Bacillales, Lactobacillales, Clos - Calanoida tridiales, Fusobacterium, Leptotrichia, Caulobacter- Surface water Deep water aceae, Neisseriaceae, and Pseudomonadales (Fig. 3). PC2 – Percent variation explained 25.46% Conversely, a few taxa of the bacteria were specific to the ambient water. Ambient water-specific bacte- ria at the genus level belonged to the Acidimicro- PC1 – Percent variation explained 42.63% biales, Flammeovirgaceae, Cryomorphaceae, Cysto- Fig. 4. Principal coordinates analysis (PCoA) of copepod- bacterineae, Alteromonas, Coxiella, Pseudospirillum, associated and ambient-water bacteria from individual sam- Thiothrix, and Mariprofundus (Fig. 3). ples. Bacterial communities from the same group of samples are represented by the same color Most of the bacterial taxa were present in both the ambient water and associated with copepods; how- ever, their contribution to the respective bacterial separated from the rest of the communities (Fig. 4) community differed among the 2 contrasting environ- and had a lower bacterial diversity as compared to ments (Fig. 3). Although our data were analyzed with the other Cyclopoida samples (Table 1). These 2 2 different databases (SILVA and Greengenes), the Cyclopoida-associated bacterial communities were phylogenetic affiliation of the bacterial 16S rRNA significantly different from the other Cyclopoida- and gene obtained with both databases was comparable. Calanoida-associated communities (p < 0.001 Bonfer- The results from the BLAST hit-score using the NCBI roni corrected), and correspond to the communities taxonomy of the 2 different databases (see Fig. S3 in associated with Oncaeidae and Lubbockiidae col- the Supplement) were significantly correlated (p < lected at Stn 4 as shown in Fig. 2. 0.01, r2 = 0.94) with a slope close to unity. The only re- The number of OTUs shared between the bacterial markable discrepancies between the 2 databases communities of the 2 depth layers and the copepod were detected for Actinomycetales and Rickettsiales, orders is indicated in Fig. 5. The 2 different copepod probably due to the lower number of sequences from orders shared 191 (8.3%) OTUs while the 2 depth lay- these groups available in the SILVA database as com- ers (100 m and 750 m) shared only 84 (3.7%) OTUs. pared to the Greengenes database (Fig. S3). The bac- Therefore, the number of shared OTUs was higher terial community composition of the ambient water within the copepod-associated than within am bient- and that associated with copepods were significantly water bacterial communities. Only 33 (1.5%) OTUs different (Unifrac significance test, p < 0.001, Bonfer- were ubiquitously present, i.e. in all 4 sample cate- roni corrected). The PCoA clearly separated ambient- gories. These ubiquitously present OTUs consisted of water and copepod-associated bacterial communities the most abundant ambient-water OTUs such as (Fig. 4), with the first coordinate accounting for 42.6% SAR11, SAR324, Chloroflexi, Desulfo bacterales, Rho - and the second for 25.5% of the samples’ variance. dobacteraceae, and Synechococcophycideae. Fur- Furthermore, the bacterial communities of the ambient thermore, the bacterial communities from the ambient water clustered according to depth, and the copepod- water and Cyclopoida-associated samples harbored a associated bacteria according to the copepod order, larger number of unique OTUs (510 [22.5%] OTUs at but with a higher variability than the ambient-water 100 m depth, 540 [23.9%] OTUs at 750 m depth, 676 bacterial communities (Fig. 4). In particular, the bac- [29.9%] OTUs in Cyclopoida) than the community terial communities of 2 Cyclopoida samples were well associated with Calanoida (241 OTUs, 10.6%). De Corte et al.: Links between copepods and bacteria 223
Gammaproteobacteria and Firmicutes (genus Bacil- CYCLO CALA lus) in partial agreement with our finding, consider- 676 241 ing the limited resolution of fingerprinting tech- (29.9%) (10.6%) 141 niques such as DGGE. 12 (6.2%) 17 The limited amount of data available and the dif- SURFACE (0.5%) (0.8%) DEEP 510 540 ferent methods used to determine the zooplankton- 7 (22.5%) 10 (23.9%) associated bacterial community composition preclude (0.3%) (0.4%) a thorough assessment of compositional differences in 33 bacterial communities between zooplankton species 224(1.5%) (0.1%) (1.1%) or different oceanic provinces. However, the available 2 14 data indicate considerable interspecific variability in (0.1%) (0.6%) the composition of the zooplankton-associated bacter- 35 ial community (Tang et al. 2010). This microbial com- (1.5%) munity is mainly associated with the exoskeleton and gut, which provide a favorable environment for bacte- Fig. 5. Venn diagram showing the shared and unique bacte- rial attachment and growth (Nagasawa & Nemoto rial operational taxonomic units (OTUs; 97% identity) and 1988, Pruzzo et al. 1996, Carman & Dobbs 1997). their relative contribution (in %) in copepods (CALA: Cala - noida; CYCLO: Cyclopoida) and ambient water (Surface and Deep) Diversity and taxonomic composition of copepod-associated bacteria DISCUSSION Previous studies indicated that the bacterial com- In this study, we used a 454 high-throughput se - munity associated with the gut of crustacean zoo- quencing approach to characterize the bacterial com- plankton consists of 2 different bacterial communi- munity associated with 2 orders of copepods and to ties: the resident bacteria persistently living in the compare them to the bacterioplankton community of gut and hence representing the stable component of the ambient water collected at the same location. the gut community, and the transient bacteria repre- Generally, the bacterial community composition ob - senting the variable gut community just passing tained in this study with 454 pyrosequencing differed through the digestive system of the host (Harris 1993, from those reported in other marine and freshwater Tang et al. 2010). In our study, some bacterial phylo- zooplankton studies using different techniques such types were consistently and abundantly found asso- as cloning and sequencing, agar plating, and CARD- ciated with the copepods and absent or only present FISH (Sochard et al. 1979, Hansen & Bech 1996, Peter in low abundances in the surrounding water (Fig. 3). & Sommaruga 2008, Grossart et al. 2009, Tang et al. However, some taxa of the copepod-associated bac- 2010, Freese & Schink 2011). These differences are teria varied considerably in abundance between the likely attributable to the different approaches used individual copepod samples, particularly in 2 out of to investigate the bacterial community associated the 9 copepod samples (Fig. 2, Table 2). These 2 with the zooplankton. Early studies on zooplankton- Cyclopoida-associated bacterial communities devi- associated bacteria used agar-plating approaches ated substantially from the community composition and consequently underestimated bacterial diversity of the other 7 samples of copepod-associated bacteria (Sochard et al. 1979, Hansen & Bech 1996). However, and were characterized by a very low abundance of the bacterial community associated with copepods chloroplasts (0.5% of total sequences; Fig. 2a). This obtained with the pyrosequencing technique was may suggest that the gut was empty at the moment comparable to a study conducted on freshwater zoo- of the extraction of the sample. These pronounced plankton-associated bacterial communities using differences between the rather similar bacterial denaturing gradient gel electrophoresis (DGGE) community composition of the 2 Calanoida and 5 combined with sequencing (Grossart et al. 2009, Cyclopoida samples, on the one hand, and the 2 Tang et al. 2010). In the latter study, the bacterial Betaproteobacteria-dominated communities in the community associated with Thermocyclops oitho - other 2 Cyclopoida samples, on the other hand, might noides (a marine and brackish cyclopoid copepod) reflect differences between the bacterial community was dominated by Betaproteobacteria, Bacteroi - composition with a filled gut (resident plus transient detes, and Actinobacteria, followed by Alpha-, and bacterial community) and after gut evacuation (resi- 224 Aquat Microb Ecol 72: 215–225, 2014
dent bacteria; Grossart et al. 2009). After gut evacua- related to the ingestion and egestion of the food. tion, only the resident bacteria adapted to live and The development of the resident copepod-associated persist in the short gut of the copepods are detectable bac terial community is likely governed by the (Grossart et al. 2009, Tang et al. 2010). An alternative specific microenvironmental conditions in copepods. explanation for these pronounced differences in the The extent to which the transient, and particularly bacterial community composition between the 2 Cy - the resident, copepod-associated bacterial communi- clopoida samples and the other zooplankton samples ties vary in their composition due to the quality of might be due to differences in the food sources, phys- food sources and periodicity in feeding activity re- iological state, or environmental conditions to which mains to be shown. the zooplankton were exposed prior to sampling. Intriguingly, we did not obtain Vibrio spp. se quen - ces in our copepod samples, in contrast to other stud- CONCLUSION ies that analyzed copepod-associated pathogens in costal and estuarine environments (Huq et al. 1983, We found significant differences between bacterial Heidelberg et al. 2002, Vezzulli et al. 2010). Although communities associated with copepods and those of Vibrio spp. play an important role in the mineraliza- the ambient water. However, our data suggest a dy- tion of chitin (Huq et al. 1983, Bassler et al. 1991, Hei- namic linkage between these 2 communities. This in- delberg et al. 2002) and account for a significant pro- teraction most likely affects the copepod- associated portion of the zooplankton-associated microbial bacterial activity and diversity. Moreover, the bacterial community (Huq et al. 1983, Heidelberg et al. 2002), diversity associated with zooplankton greatly di- their role in the open ocean zooplankton remains verged in 2 out of 9 samples, with specific phyloge- unknown. netic groups dominating in these 2 samples, suggest- ing that the food sources and feeding status of the zooplankton might influence the bacterial community Comparison between the copepod-associated and composition associated with the guts of copepods. the ambient-water bacterial communities
Grossart et al. (2009) suggested that the diversity of Acknowledgements. We thank the captain and crew of the RV ‘Pelagia’ for their support and splendid atmosphere on bacteria associated with zooplankton is mainly de - board. E.S. was supported by the ESF EuroCores project pendent on host−symbiont interactions, food, and the MOCA financed via the Austria Science Fund (FWF, project ambient bacterial community to which the host is number I 486-B09) and the FWF project MICRO-ACT (pro- exposed. We compared the copepod-associated bac- ject number 23234-B11). Analytical work was supported by teria to the bacterioplankton to determine the link- the FWF project Z194-B17 to G.J.H. and by the Marie Curie Fellowship (PIEF-GA-2011-299860) to D.D.C. We thank Jef- age between the 2 bacterial communities. Despite ferson Turner for his comments and the English editing of the significant differences between the copepod- the manuscript. associated and the ambient-water bacterial com - munities (Figs. 2 & 3), the presence of shared OTUs LITERATURE CITED (Fig. 5) between all the samples (1.5% of the total OTUs) suggests a limited exchange of bacteria be - Alldredge AL (2000) Interstitial dissolved organic carbon tween the ambient water and the copepods. (DOC) concentrations within sinking marine aggregates The long tail of rare OTUs obtained for the ambient and their potential contribution to carbon flux. Limnol water in the rank-frequency distribution (Fig. S2) Oceanogr 45: 1245−1253 Alldredge AL, Cohen Y (1987) Can microscale chemical might provide a seed-bank of OTUs adapted to envi- patches persist in the sea? Microelectrode study of mar- ronmental conditions different from those prevailing ine snow, fecal pellets. Science 235:689−691 in the ambient water (Pedrós-Alió 2006). However, Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, we were not able to detect OTUs of the resident Miller W, Lipman DJ (1997) Gapped BLAST and PSI- BLAST: a new generation of protein database search copepod-associated bacterial community in the am- programs. Nucleic Acids Res 25: 3389−3402 bient water, most likely because of the insufficient Azam F, Fenchel T, Field JG, Gray JS, Meyer-Reil LA, coverage of the ambient-water community (Fig. 1). Thingstad F (1983) The ecological role of water-column Taken together, our findings point toward a dyna - microbes in the sea. Mar Ecol Prog Ser 10:257−263 Bassler BL, Yu C, Lee YC, Roseman S (1991) Chitin utiliza- mic relationship between bacteria, zooplankton, and tion by marine-bacteria — degradation and catabolism of the environment where the dispersal of the copepod- chitin oligosaccharides by Vibrio furnissii. J Biol Chem associated transient bacterial community is mainly 266: 24276−24286 De Corte et al.: Links between copepods and bacteria 225
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Editorial responsibility: Fereidoun Rassoulzadegan, Submitted: October 29, 2013; Accepted: March 25, 2014 Villefranche-sur-Mer, France Proofs received from author(s): May 9, 2014