Bacterial Diversity in Ships' Ballast Water, Ballast-Water Exchange

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Bacterial diversity in ships’ ballast water, ballast-water exchange, and implications for ship-mediated dispersal of microorganisms Despoina S. Lymperopoulou, and Fred C. Dobbs Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03108 • Publication Date (Web): 30 Jan 2017 Downloaded from http://pubs.acs.org on February 3, 2017

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Environmental Science & Technology

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Bacterial diversity in ships’ ballast water, ballast-water exchange, and implications for

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ship-mediated dispersal of microorganisms

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Despoina S. Lymperopoulou† and Fred C. Dobbs‡,*

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†Department of Plant and Microbial Biology, University of California – Berkeley, 331 Koshland Hall, Berkeley, California 94720 USA

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‡Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, 4600 Elkhorn Avenue, Norfolk, Virginia 23529 USA

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Corresponding author

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*Phone: (757) 683-5329; Fax: (757) 683-5303; Email: [email protected]

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Abstract

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Using next-generation DNA sequencing of the 16S rRNA gene, we analyzed the composition

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and diversity of bacterial assemblages in ballast water from tanks of 17 commercial ships

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arriving to Hampton Roads, Virginia (USA) following voyages in the North Atlantic Ocean.

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Amplicon sequencing analysis showed the heterogeneous assemblages were: 1) dominated by

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Alpha- and Gammaproteobacteria, Bacteroidetes, and unclassified Bacteria; 2) temporally

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distinct (June vs. August/September); 3) highly fidelitous among replicate samples. Whether

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tanks were exchanged at sea or not, their bacterial assemblages differed from those of local,

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coastal water. Compositional data suggested at-sea exchange did not fully flush coastal Bacteria

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from all tanks; there were several instances of a genetic geographic signal. Quantitative PCR

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yielded no Escherichia coli and few instances of Vibrio species. Salinity, but not ballast-water

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age or temperature, contributed significantly to bacterial diversity. Whether anthropogenic

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mixing of marine Bacteria re-structures their biogeography remains to be tested.

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Keywords: ballast water, Bacteria, Illumina sequencing, 16S rRNA, ballast-water exchange

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Introduction

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Ballasting operations associated with commercial shipping translocate enormous volumes of

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water daily, nearly all of which, except an un-pumpable residual, eventually is discharged, often

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in coastal areas and navigable inland waters. In the United States alone, more than 100 million

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metric tonnes of overseas ballast water is discharged annually.1 Entrained in those discharges are

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organisms taken up when ballasting and which survived days to months in the tanks. In this way,

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ballasting operations have introduced nonindigenous organisms globally, and some have

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subsequently flourished in novel environments and are implicated in deleterious economic,

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environmental, and public-health issues.2, 3 The case for such invasions is much better articulated

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for invertebrates and harmful algae than for Bacteria,4 but there is strong evidence that ballast-

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water discharges introduce fecal-indicator bacteria5-7 and bacterial pathogens.8-14

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While pathogens certainly are a concern with respect to human and ecosystem health, their

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abundance in ballast water, as in natural aquatic environments, is very low compared to

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environmental Bacteria. Indeed, in the absence of treatment to kill or remove microorganisms,

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overseas ballast water discharged in the United States delivers 107 to 109 bacteria cells/L,15-19

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yielding on order of 1019 cells per year; approximately the same total was estimated for ballast

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discharges in Canada.18 Prior to application of next-generation sequencing approaches, scientific

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focus on these astronomical numbers of cells was principally, but not exclusively,5, 20 to

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enumerate them using epifluorescence microscopy, a tool incapable of assessing their

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community composition and diversity. Such enumerative studies included samples from ends of

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ships’ voyages,15, 16, 18, 21 while others examined the temporal dynamics of bacterial numbers

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during voyages.17, 19, 22-24



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Given concerns about conveyance and release of nonindigenous or pathogenic

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microorganisms and larger organisms in the course of ballasting operations, some countries

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regulate the maximum concentrations of organisms allowed in discharged ballast water.25 Global

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standards were proposed in the International Maritime Organization’s “Convention for the

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Control and Management of Ships’ Ballast Water and Sediments” (http://www.imo.org), which

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will enter into force in 2017. Although there is a considerable industry in developing technology-

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based treatments to reduce the concentration of discharged organisms,26 the interim practice is to

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flush coastal water from ballast tanks and replace it with open-ocean water, so called ballast-

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water exchange (BWE). The thought is first, to discharge coastal organisms and second, to

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ensure ballast water released subsequently contains open-ocean organisms unable to survive and

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reproduce in a coastal or freshwater environment.27

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Little is known about the composition and diversity of Bacteria in ballast water, in contrast to

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our understanding of its taxonomic diversity of metazoan species,28-30 eukaryotic

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phytoplankton,17, 31, 32 and other protists.6, 33 The modern standard for assessing microbial

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diversity is high throughput sequencing, an approach based in molecular biology and one that

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does not involve cultivation, but instead employs DNA sequencing to provide a much more

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comprehensive view of microbial assemblages. Following a call3 for this technique to be applied

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to microorganisms in ballast water, such studies recently began to appear.14, 34-40 In the present

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study, we used next-generation sequencing (NGS) of the 16S rRNA gene to characterize Bacteria

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in ballast water collected from 17 commercial vessels arriving to Hampton Roads, Virginia,

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USA, following voyages in the North Atlantic Ocean. Based on genetic signatures, we describe

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assemblages of Bacteria from ballast tanks and compare them, whether exchanged at sea or not,

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with one another, with bacterial communities of local waters, and with previous studies. We


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hypothesized we would detect compositional differences with respect to ships’ previous ports of

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call, whether the ships had exchanged at sea, and the length of time water had been held. We

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conclude by considering the implications of this research on the distribution of aquatic

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microorganisms.

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Materials and Methods

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Ethics statement: Ballast-water samples were collected by colleagues from the Smithsonian

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Environmental Research Center (SERC), which routinely samples vessels as part of a joint

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program with the US Coast Guard. Terms of agreement with ships’ owners and companies

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operating vessels prohibit our publishing names of vessels. Code names were assigned to vessels

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by SERC scientists.

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Sample collection and processing: The port system at Hampton Roads, Virginia, includes ports

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in Norfolk, Portsmouth, and Newport News on the Elizabeth and James Rivers, tributaries close

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by the mouth of Chesapeake Bay, largest estuary in the United States. Coal exports and

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containers are the principal traffic, with nearly 79 million short tons in 2013, placing Hampton

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Roads as the 6th largest port in the US, and 5th largest with respect to total foreign trade

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(http://www.aapa-ports.org/). From 2011 to 2103, the National Ballast Information

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Clearinghouse ranked the port third in the United States with respect to discharge of overseas

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ballast water (40.3 million m3, http://invasions.si.edu/nbic/search.html).

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Ballast water was sampled from 17 vessels, 10 in June 2013 and 7 in August/September 2013

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(Table 1). Twelve ships had their last port of call in Europe and had conducted BWE in tanks

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from which samples were collected (Table 1). The last ports of call of the remaining 5 ships were



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in North America; two of these (CB21 and CB25) had exchanged ballast water at sea. Of those

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tanks that underwent BWE, exchange volumes were ≥ 300% in most cases, except for CB24,

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CB37, CB39, and CB40, for which exchange volumes were approximately 100%. The remaining

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ships (CB26, CB28, CB29) did not exchange at sea as their voyages remained within the US

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Exclusive Economic Zone, and were in compliance with US statutes in this regard.

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From the upper-water column in one tank on each vessel, five independent, replicate, 1-L

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water samples were collected into acid-washed and autoclaved 1L polyethylene Nalgene®

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bottles, for a total of 85 1-L samples. Because each ship is represented by samples from one

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ballast tank only, reference in this report to a “tank” uniquely ties it to a particular “vessel” or

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“ship”, and vice-versa. Temperature and salinity were measured in situ with a portable meter

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(YSI Inc., Yellow Springs, OH, USA). Samples were kept in the dark on ice for transport to the

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laboratory, usually within 1 hour, then two 500-ml aliquots from each bottle were separately

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filtered (to reduce filtration time) through polyethersulfone filters (0.2 µm pore size, Supor 200

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Membrane Disc Filters, Cat. #60301, PALL, Cortland, NY, USA) and the filters were stored at -

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20°C. In June 2013, water samples were collected from two nearby locations for comparison,

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one set from Old Dominion University’s Sailing Pier (ODUSP) on the Elizabeth River, 8.9 km

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upriver from the Norfolk harbor. The other local water was collected at Norfolk’s Yacht and

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Country Club (YC) on the Lafayette River, at its junction with the Elizabeth River and

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approximately 11 km upriver from the harbor (n=5 1-L samples per location). We did not take

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samples from the port proper, because we did not know whether any part of the port represents

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undiluted “local water”.

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DNA extraction and sequencing: DNA was extracted with PowerSoil® DNA Isolation Kit

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(Cat. #12888, Mo Bio, Carlsbad, CA, USA) according to manufacturer’s instructions, except that 6 ACS Paragon Plus Environment

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the kit’s PowerBead tubes were replaced with 15ml Falcon tubes. Whether ballast water or local

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water, two filters representing a bottle’s 1-L volume were combined in a single tube. DNA was

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separately extracted from three replicate samples, representing a total of 3 L of water. Filters

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representing the other two replicates were archived at -80°C. Thus, 57 samples were extracted,

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51 of ballast water and 6 of local water. Checks of DNA yield and purity, and subsequent

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amplification and sequencing of the V4 and V5 variable region of the 16S rRNA gene with

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MiSeq Illumina technology, are detailed in the Supplementary Information.

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Sequence processing: Details are given in the Supplementary information. Briefly, we

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processed raw Illumina sequence reads using mothur v.1.33.3.41 After trimming primer

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sequences and barcodes, the two data sets (June and August/September) were combined,

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dereplicated, and unique sequences were aligned against the SILVA reference database 10242

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and further corrected for erroneous sequences by pre-clustering them.43, 44 High-quality

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sequences were classified using the Ribosomal Database Project Naïve Bayesian Classifier45 and

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unspecific PCR amplicons (mitochondria, chloroplasts, Archaea, Eukarya, unknown domain)

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were removed. The remaining sequences were clustered into operational taxonomic units (OTUs)

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at sequence divergences of 3%.46 Finally, singleton sequence OTUs were removed. To normalize

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sequencing effort across samples without compromising their measures of genetic diversity, all

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samples (except one) were randomly subsampled to the number of reads in the sample with the

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second fewest reads (13,075 sequences, CB36.R2). A sample with the least reads (7,243

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sequences, CB36.R4) was intentionally removed from the analysis. The sequence data were

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deposited in the GenBank Short Read Archive under accession number SRP045433.

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Bacterial abundance: Values were estimated by quantifying 16S rRNA gene copy numbers

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using quantitative PCR (qPCR) to amplify primers 341F and 534R.47 Each replicate sample 7 ACS Paragon Plus Environment


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(n=57, i.e., (17 tanks + 2 local sites) X 3 replicates per tank or site) was measured in duplicate

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and no-template controls were included. Details are in the Supplementary Information.

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Species–specific qPCR: To detect Bacteria of public-health concern, we quantified Escherichia

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coli (E. coli), Vibrio parahaemolyticus (V. parahaemolyticus), and V. vulnificus using

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fluorophore-based detection of uidA,48 vvhA,49 and tlh genes.50 For V. cholerae, quantification

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was based on a SYBR Green I assay during amplification of a sequence of the intergenic spacer

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region between the 16S rRNA and 23S rRNA.51 Details are in the Supplementary Information.

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Statistical analyses: Rarefaction curves were calculated in mothur v.1.33.341 with replicate

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CB36.R4 removed (see above). Alpha diversity estimators (Shannon’s H’; Simpson index of

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diversity (1-D); and the richness estimator SChao1) were calculated in PAST 2.17c software.52

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Sequence abundances were square-root transformed to diminish the effect of dominant OTUs

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while accounting for less dominant ones, then cluster analysis was performed with the Bray-

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Curtis index at 1,000 bootstrap values, to graphically illustrate the relationships among the

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different samples based on the UPGMA algorithm in PAST. Distance matrices between samples

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were calculated using the Bray–Curtis index in PAST and used for nonmetric multidimensional

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scaling (NMDS) and Principal Components Analysis (PCA), in conjunction with physical

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variables including salinity, temperature, and ballast-water age (defined as the number of days

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since the most recent loading of water in the tank). All OTUs’ abundance data were used in the

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NMDS, while the PCA was constructed based on phyla (and classes within Proteobacteria)

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frequencies. These distance matrices were also used in analysis of molecular variance

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(AMOVA), to examine differences in bacterial assemblages among vessels that performed BWE,

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vessels that did not, and local samples, and between all possible pairwise combinations.

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AMOVA was performed in mothur. To assess the explanatory power of BWE and ballast water 8 ACS Paragon Plus Environment

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(BW) age on the community composition and membership, we used a permutational multivariate

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analysis of variance (PERMANOVA) with the Bray-Curtis index and based on 1,000

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permutations implemented by ADONIS test in the vegan package53 in R.

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Separately for tanks exchanged at sea (n=14) and for all tanks (n=17), we regressed

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dependent variables species (OTU) richness, observed (S) and expected (SChao1), on independent

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variables salinity, temperature, and ballast-water age. A Kolmogorov-Smirnoff statistic was

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performed to test normality of these data. As many were not normal, regressions were tested for

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significance at α = 0.05 through randomization of data (10,000 iterations/regression) to generate

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customized F distributions from which p values were calculated.

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Results

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Salinity, temperature, and age of ballast water: In the 14 tanks exchanged at sea, salinities

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ranged from 33.7 (CB23) to 37.0 (CB18, CB42) (Table 1), consistent with open-ocean salinities.

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Water in the 3 unexchanged tanks reflected a lower salinity, in keeping with coastal water from

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Massachusetts (CB26, salinity 31.0) and Mobile Bay, Alabama (CB28, CB29, salinities 20.1 and

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20.2, respectively). Overall, however, there was no difference in mean salinity between sampling

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periods (June, 32.07; August/September, 35.66; t-test, unequal variance, two-sided, p>0.05).

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Salinities of local water samples were 24.0 (ODUSP) and 19.8 (YC). Across all vessels, ballast

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water had been onboard 2 days (CB21) to 28 days (CB29) and ballast-water temperatures on

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collection ranged from 20.0°C (CB21) to 32.0°C (CB41). Mean water temperatures in June were

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less than those in August/September (24.8º vs. 27.9º, t-test, equal variance, two-sided, p5% of

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relative abundance only in CB25 (18%) (Figure S2B). Among Gammaproteobacteria, the group

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having greatest relative abundance among vessels was the order Thiotrichales (4.5%), while

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Vibrionales demonstrated low relative abundances (0.01% to 1%). Epsilonproteobacteria were

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rarely retrieved, but in one case (CB18), Campylobacterales accounted for 8% of OTUs, mainly



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represented by Sulfuromonas sp. Within Bacteroidetes, Flavobacteriales dominated with relative

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abundances as high as 38% (CB24).

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Thirty seven OTUs, representing > 0.5% of the sequences for each higher taxonomic group

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(phylum), accounted for 50% of the total sequences (Figure S3). The most abundant OTU

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(OTU1, 10% of all sequences) remained unaffiliated at the confidence level of 80% we used.

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After manual classification, we assigned OTU1 to the SAR11 clade based on phylogenetic

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analysis (Figure S4). OTU1 formed a distinct monophyletic group within Alphaproteobacteria

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that also included OTU13 and was closely related to Pelagibacter and SAR11. This coherent

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phylogenetic cluster ranged from 1.2% (CB28, low-salinity water from Mobile Bay) to 19.9%

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(CB36, exchanged at sea, tied for highest salinity). OTU1 also was found in local waters, 8.5%

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in YC and 11.4% in ODUSP.

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Although many OTUs were not identified to genus, those that were represented major phyla

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and were affiliated with the genera Cycloclasticus (OTU 4; Gammaproteobacteria; range 0.1%

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(CB25, CB26) to 19% (CB37)), Novosphingobium (OTU 12; Alphaproteobacteria; 0% (CB38-

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42) to 17% (CB25)), Sulfuromonas (OTU 32; Epsilonproteobacteria; 0% (CB24, 32, 36, 39-42)

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to 8% (CB18)), Tenacibaculum (OTU 5; Flavobacteriia; 0% (CB38-42) to 13.3% (CB25)),

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Gilvibacter (OTU 19; Flavobacteriia; 0% (CB21-24, 26-32) to 10.4% (CB40)), and Formosa

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(OTU 23; Flavobacteriia; 0% (CB36-42) to 3.6% (CB27)).

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Bacterial abundance: Based on qPCR results, 16S rRNA gene numbers were greatest in local

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water samples (mean 2.30 x 109 copies/L ± SD 1.91 x 109, n=2) and least in June samples from

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tanks that had undergone BWE (5.72 X 106 ± 7.71 x 106, n=7). In exchanged tanks sampled in

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August/September (5.07 x 108 ± 4.33 x 108, n=7) and in June tanks not exchanged at sea (2.15 x

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108 ± 3.65 X 108, n=3), values were intermediate. 12 ACS Paragon Plus Environment

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Fecal indicator and pathogenic Bacteria: In sequence data, the genus Enterococcus was

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detected only at the noise level (one in CB37.R1). The genus Escherichia was represented by

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rare sequences sporadically retrieved from a few ships. Evidence of the genus Vibrio consisted of

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only three sequences and comprised about 0.3% of all reads collectively across all ships, with a

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single OTU accounting for approximately 0.25% of the vibrios detected. Local waters accounted

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for 40% of all vibrio sequences. In qPCR analyses, most samples were negative for Vibrio

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cholerae, V. vulnificus, V. parahaemolyticus, and E. coli. Exceptions were vessels CB38, CB39,

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CB40, and CB41, positive for V. parahaemolyticus with 27, 218, 50, and 121 tlh gene copies per

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100 ml of filtered water, respectively. Local waters, sampled in June only, yielded positive

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results for V. vulnificus, with 2.1 x 102 (YC) and 3.9 x 102 (ODUSP) vvhA gene copies per 100

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ml of filtered water.

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Multivariate analyses: Cluster analysis of the sequence data showed clear temporal

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demarcation of samples (Figure 2). Bacterial assemblages in tanks sampled in August/September

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had only a 27% Bray-Curtis similarity to those collected in June, as well as to local water

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samples, also collected in June. Within both times, however, the triplicate, within-tank samples

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(only duplicates for CB36) clustered together, except CB27.R1, which “misclassified” with

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samples from CB24. There was stronger within-tank similarity in August/September, from

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approximately 70% (CB36, CB37, CB38) to 78% (CB42). In June, within-tank similarity values

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ranged from about 60% to 67%, with two exceptions, CB24 (53%) and CB32 (41%). The

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Bacteria signal from the two local sites (54% similarity) first clustered (34%) with samples from

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tanks holding unexchanged, low-salinity water from Mobile Bay (CB28, CB29) to form a group

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distinct from all others. The only other samples from an unexchanged tank (CB26) contained



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coastal Massachusetts water and clustered first (49%) with samples from a tank exchanged south

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of Nova Scotia (CB21).

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Among ships that exchanged ballast water in the North Atlantic Ocean, there were instances

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in which a geographic signal arguably emerged, one assignable to ships’ last ports of call. First,

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in August/September samples, the strongest inter-vessel cluster (45%) occurred between CB39

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(Rotterdam) and CB40 (Montoir). This cluster was subsequently joined by CB38 (Ijmuiden),

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then CB41 (Rotterdam), to form a larger group (similarity 34%) of vessels having last ports of

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call either in the Netherlands or France. Second, two ships having last ports of call in southern

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Italy (CB37, CB42) formed their own cluster (38%). Third, two ships with last ports of call in

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Germany (CB23, CB27--other than replicate R1) clustered first with one another (42%).

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Results of NMDS analysis (stress: 0.17) were consistent with the cluster analysis; replicate

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samples were tightly grouped and samples collected in June were topologically separated from

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those taken in August/September (Figure 3). Both analysis of molecular variance (AMOVA) and

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Adonis test showed a significant difference among bacterial assemblages from the three groups

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(unexchanged, exchanged, and local water) and their pairwise combinations (p99%.27, 61 With respect to pathogens in particular,

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ballast water is thought by some8, 66 but not all67, 68 to have delivered an Asian strain of Vibrio

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cholerae to South America and caused the 1991 cholera epidemic. Haendiges et al. (2016)69

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speculated that ballast water introduced a non-autochthonous strain of V. parahaemolyticus to

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upper Chesapeake Bay. Brinkmeyer14 reported 60 human, fish, and plant pathogens in ballast

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water, suggesting their potential for translocation.

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Pathogens and fecal-indicator Bacteria: With respect to discharge regulations specifying

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maximum concentrations of fecal-indicator (E. coli, enterococci) and pathogenic (Vibrio

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cholerae) Bacteria, this study’s results indicated few such organisms among 17 ships. Whether

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through DNA sequence analysis or qPCR amplification of extracted DNA, these organisms were

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found, if at all, in miniscule numbers. Of other potentially pathogenic Vibrio species tested for

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with qPCR, only V. parahaemolyticus was detected in several ballast tanks. In contrast, there are

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multiple reports of ballast water containing vibrios as determined by culture, fluorescent17 ACS Paragon Plus Environment


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antibody techniques, PCR, or sequencing methods.8, 12-15, 17 Perhaps ballast water in this study

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truly was nearly devoid of these Bacteria. Another possibility, however, is that the concentration

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of DNA specific for these forms was at such low levels that they went undetected with the broad

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bacterial marker used, i.e., a portion of the 16S rRNA gene amplicon. That they were largely

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undetected with qPCR may reflect the heterogeneity of gene markers in environmental strains.

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Finally, if there was water-column heterogeneity in the distribution of these Bacteria, as has been

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shown for zooplankton,70 then any aggregations may not have been sampled, as all within-tank

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replicates were collected from a single water depth.

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Comparisons with other studies: Across the three NGS studies of Bacteria in ballast water,

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there was a 15-fold range of unique OTUs reported (Table 2), from 21,000 (present

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study). Of tanks that underwent BWE, bacterial diversity estimated by Shannon’s H’ was highest

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overall in the present study, but Simpson’s evenness index had approximately the range reported

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by Ng et al. (2015).40 With reference to aquatic species-area theory,71 the greater number of

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bacterial OTUs and higher diversity in the present study likely reflect its greater number of ships,

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samples, and volumes of water extracted. In exchanged tanks, 16S rRNA gene counts of the

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present study encompassed those of the other two and generally agree with the range of direct

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counts of bacteria reported in ballast water,15, 16, 24 except at the low end, June samples

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representing exchanged ballast (mean 5.72 x 106 gene copies/L).

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When the composition of Bacteria was compared among investigations, the relative

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dominance of Prochlorococcus- and Synechococcus-associated cyanobacterial OTUs (GPIIA

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group) and the SAR11 cluster was greatest in this study, suggesting a stronger open-ocean signal

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in the tanks following exchange. Indeed, the low salinities of samples reported by Ng et al.

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(2015),40 values from 14 to 20.7, suggest BWE occurred in coastal water. While Ng et al. 18 ACS Paragon Plus Environment

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(2015)40 found Gammaproteobacteria heavily dominated the bacterial assemblage in exchanged

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tanks, Brinkmeyer (2016)14 and the present study instead determined the Roseobacter clade

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within the Alphaproteobacteria predominated. All three studies reported Thiotrichales as

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dominant within Gammaproteobacteria and Flavobacterales among Bacteroidetes.

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We also compared our results with descriptions of bacterioplankton in oceanic surface

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waters.57, 72, 73 This comparison is not straightforward, given no coincidence of geography or

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sampling depth, that ballast tanks do not preserve open-ocean conditions and are not conducive

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to survival of oceanic microbial communities,17, 22 and the sometimes incomplete nature of

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BWE.27, 61 Nonetheless, the range of OTUs in ballast water (348 to 1,468) was within that

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reported by others for upper-ocean waters: 245 to 2,063 OTUs;57 446 to 1,157 OTUs;72; and 796

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to 1,258 OTUs.73 Comparisons of diversity are more limited; only Jing et al.73 reported

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Shannon’s H’ (mean 4.25, SD 0.06, range 4.17 to 4.31, n=3). By comparison, the present study’s

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ballast-water samples had essentially the same mean, but a greater range (mean 4.26, SD 0.52,

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range 3.20 to 5.09, n=14), a result presumably related to multiple combinations of ballast source

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water, tank conditions, and water age.

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With respect to major taxa, Proteobacteria (mostly Alpha- and Gamma-) dominated ballast-

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tank assemblages, as they do those of oceanic surface water, but usually in lesser proportions

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within tanks (21% to 58%; CB42 highest at 72%) compared to those reported previously, about

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88% in surface waters, n=6,57 and 61% to 68%, n=3.73 We attribute this differential in part to the

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comparatively high proportion of unclassified Bacteria in our analysis, as well as finding high

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proportions of Bacteroidetes and Planctomycetes in some tanks. Consistent with global ocean

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surveys,74, 75 we found SAR11 and GPIIA group–associated cyanobacterial OTUs (e.g., genera

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of Prochlorococcus and Synechococcus) as dominant taxa in exchanged tanks. 19 ACS Paragon Plus Environment


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Indeed, we assigned OTU1, the most abundant taxon in tanks, to the SAR11 clade of

417

Alphaproteobacteria, which comprises about one third of Bacteria in multiple locations of the

418

open ocean.76, 77 It is noteworthy that this dominance was exhibited even in the oldest ballast-

419

water sampled (CB32, 26 days, 10.6% relative dominance). The success of this taxon has been

420

attributed to efficient genome streamlining, allowing it to adapt to nutrient-poor environments.74

421

We do not know, however, whether presumably nutrient-poor conditions in ballast tanks sustain

422

predominance of SAR11.

423

Oceanic OTUs affiliated with Rhodobacteraceae and specifically Roseobacter clade-

424

affiliated (RCA) ones, e.g. Roseobacter and Sulfitobacter, also were ubiquitous and abundant in

425

tanks. RCAs are cosmopolitan in temperate seawaters.78 Sulfitobacter shows close association

426

with organic particles, especially diatoms,79 suggesting the group is intrinsic to fecal pellets.80

427

Members of the RCA cluster metabolize dimethylsulfoniopropionate (DMSP), the major source

428

of organic sulfur in the world’s oceans, and have been associated with DMSP-producing

429

dinoflagellates.81 Therefore, their prevalence suggests the presence of such algae, some of which

430

are harmful. Algal OTUs affiliated with Prorocentrum spp. and Alexandrium spp. have been

431

reported from ballast tanks.38

432

OTU2 (Saprospiraceae) was particularly pronounced in CB23, CB24, and CB27; in the latter

433

two, it was more abundant than OTU1. OTU2, along with other abundant OTUs (≥0.5%)

434

affiliated with Bacteroidetes, represents a phylum abundant in coastal waters and in the oceans. It

435

is assumed its members attach to particles and degrade polymers, because they exhibit a positive

436

correlation to phytoplankton blooms82 and organic-matter particles.83 OTU4 predominated in

437

tanks on CB37 and CB40 and is affiliated with Cycloclasticus spp., a bacterium mainly found in

438

marine sediments and linked to degradation of toxic polycyclic aromatic hydrocarbons (PAH).84 20 ACS Paragon Plus Environment

Page 21 of 41


439

Another presumed PAH degrader, OTU12 (Novosphingobium spp.-affiliated),85 dominated

440

exclusively in CB25. Presence of these PAH degraders suggest residual coastal water in the

441

tanks.14

442

Cyanobacteria were abundant in local waters, especially OTU3 and OTU1, but usually

443

comprised a smaller fraction of bacterial relative abundance in ballast water, albeit larger ones

444

than those reported by others.14, 40 In particular, CB21 and CB29 contained 10% and 12%

445

Cyanobacteria, respectively, values that might be underestimates. In a test of ballast-water

446

treatment using propidium mono-azide to differentiate between living and dead cells, Fujimoto et

447

al. (2014)34 suggested the number of cyanobacterial 16S rRNA gene sequences in intake waters

448

was masked by DNA in dead or dying cells, also known as “relic DNA”.86

449 450

Supporting Information

451

Additional “Materials and Methods” and Figures S1-S6 and Tables S1-S2. This information is

452

available free of charge via the Internet at http://pubs.acs.org.

453 454

Acknowledgements

455

This research was supported in part by the U.S. National Science Foundation’s Emerging

456

Infectious Disease program [Grant EF-0914429]. We appreciate reference strains of bacteria

457

from Profs. Rita Colwell and Henry Neal Williams, the assistance of Mr. Patrick Tennis and

458

Prof. Alexander Bochdansky with regressions, and comments from anonymous reviewers. We

459

especially thank colleagues from the SERC who sampled ballast tanks and helped interpret

460

ships’ documents: Drs. Kimberly Holzer and K. Jenny Carney, and Ms. Danielle Verna. 21 ACS Paragon Plus Environment


Page 22 of 41

461

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Pedrotti, M.; Beauvais, S.; Kerros, M.-E.; Iversen, K.; Peters, F. Bacterial colonization of

Dyksterhouse, S. E.; Gray, J. P.; Herwig, R. P.; Lara, J. C.; Staley, J. T. Cycloclasticus


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690

85.

Notomista, E.; Pennacchio, F.; Cafaro, V.; Smaldone, G.; Izzo, V.; Troncone, L.;

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Varcamonti, M.; Di Donato, A. The marine isolate Novosphingobium sp. PP1Y shows specific

692

adaptation to use the aromatic fraction of fuels as the sole carbon and energy source. Microb.

693

Ecol. 2011, 61, (3), 582-594.

694

86.

695

DNA is abundant in soil and obscures estimates of soil microbial diversity. bioRxiv 2016,

696

043372.

Carini, P.; Marsden, P. J.; Leff, J. W.; Morgan, E. E.; Strickland, M. S.; Fierer, N. Relic

697



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Table 1. Designations and characteristics of local water samples and ballast water samples collected from vessels arriving to Hampton Roads, Virginia, USA, in June or August/September 2013. Management = percentage of the tank’s volume exchanged in the open ocean; all exchanges ≥300% were flow-through, all exchanges approximately. 100% were empty-refill; Age = (days between BWE exchange and sampling); Volume = volume of ballast tank; BWE = location of BWE initiation; N/A = not applicable. Vessels CB26, CB28, and CB29 did not exchange ballast at sea as their voyages did not extend beyond the US Exclusive Economic Zone. Samples

Vessel SERC ID

Jun 2013 1 CB18 2 CB21 3 CB23 4 CB24 5 CB25 6 CB26 7 CB27 8 CB28 9 CB29 10 CB32 Aug/Sep 2013 11 CB36 12 CB37 13 CB38 14 CB39 15 CB40 16 CB41 17 CB42 Local Samples 18 ODUSP 19 YC

Management (% Exchange)

Temperature (°C)

338 300 307 100 354 No exchange 342 No exchange No exchange 324

25.0 20.0 27.0 25.0 26.5 24.0 24.5 24.0 27.0 25.0

300 100 342 103 99 371 312 N/A N/A

Salinity

BWE Lat. (W) Long. (N)

Age (days)

Volume (m3)

37.0 35.1 33.7 35.7 35.9 31.0 35.4 20.1 20.2 36.6

14 2 6 10 4 7 13 16 28 26

3109 1463 3736 482 1206 1751 5317 1371 1593 5082

Moneypoint, Ireland Canso, Canada Hamburg, Germany Ghent, Belgium Port Alfred, Canada Somerset, MA Stade, Germany Mobile, AL Mobile, AL Brindisi, Italy

52.23 41.23 43.81 49.65 38.65

21.48 65.20 43.00 14.87 59.83

50.47

22.25

41.35

36.53

27.0 25.0 26.0 30.0 27.0 32.0 28.0

35.1 36.3 35.6 35.8 34.6 35.2 37.0

12 10 9 10 13 14 9

1514 1263 2119 5595 622 1011 2425

Brake, Germany Taranto, Italy Ijmuiden, Netherlands Rotterdam, Netherlands Montoir, France Rotterdam, Netherlands Brindisi, Italy

43.30 37.77 46.92 47.20 44.82 47.61 36.43

16.27 15.08 23.43 18.39 79.76 36.96 37.05

26.2 25.0

24.0 19.8

N/A N/A

N/A N/A

N/A N/A

N/A N/A

N/A N/A

Last Port of Call


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Table 2. Comparison of three studies that have used next-generation DNA sequencing and subsequent metagenomic analysis to evaluate composition and diversity of Bacteria in ships’ ballast water. Ng et al. (2015)40

Brinkmeyer (2016)14

this study

3

5

17

volume extracted per sample (L)

2.4 a

approx. 5 b

3

sample replication

none

none

3 replicates/sample

age of water (days)

4 to 107

10 to 30

2 to 28

Roche 454

Ion Torrent

MiSeq Illumina

1,395

not given

21,371

Shannon H’ exchanged tanks

2.67 to 4.32; n=3

2.45 to 3.99; n=4

3.20 to 5.09; n=14

Simpson index exchanged tanks

0.71 to 0.95; n=3

not given

0.87 to 0.98; n=14

1.2 to 3.7 x 108; n=4

5.72 x 106 to 5.07 x 108; n=14

number of tanks sampled

NGS technology unique OTUs

16S rRNA gene counts/L 5 x 107 to 8 x 108 c; n=3 exchanged tanks a

DNA was extracted from 20 ml of concentrated water sample (equivalent to 2.4 L of original water sample)

b

pers. comm.

c

estimated from their Figure 1



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Figure legends Figure 1. Relative abundances of partial (approximately 260 bp) sequences of bacterial 16S rRNA gene were estimated by classification at the phylum level, using MOTHUR with a modified 16S rRNA database from the Ribosomal Database Project. The diverse phylum of Proteobacteria is represented at the class level with different shades of green. A vertical line separates the ballast water samples (CB labels) from those of local Norfolk waters (ODUSP and YC). Key for symbols beneath sample labels: Black triangles=local Norfolk water samples; Squares=tanks sampled in June; Dots=tanks sampled in August/September; Black, gray, or red fill=tanks that underwent a 300% (or greater) volume exchange, a 100% volume exchange, or no exchange, respectively. Un. Proteo=unclassified Proteobacteria.

Figure 2. Cluster diagram for the 19 samples (3 replicates/sample, except for CB36, with only 2 replicates) constructed from a Bray-Curtis similarity matrix of square-root transformed OTU abundances. One thousand bootstrap analyses were conducted. Sample designations: Black triangles=local Norfolk water samples; Squares=tanks sampled in June; Dots=tanks sampled in August/September; Black, gray, or red font and fill=tanks that underwent a 300% (or greater) volume exchange, a 100% volume exchange, or no exchange, respectively. Country (and for USA samples, state) of last port of call indicated: B (Belgium); CDN (Canada); D (Germany); FRA (France); I (Italy); IRL (Ireland); NET (Netherlands); USA-MA (Massachusetts); USA-AL (Alabama). Local water samples indicated as USA-VA (Virginia).


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Figure 3. Non-metric multidimensional (NMDS) scaling plots in two dimensions constructed from a Bray-Curtis matrix of square-root transformed OTU abundances, including environmental variables (ballast water age, salinity, and temperature). Sample designations: Black triangles=local Norfolk water samples; Squares=tanks sampled in June; Dots=tanks sampled August/September; Black, gray, or red fill=tanks that underwent a 300% volume exchange, a 100% volume exchange, or no exchange, respectively.



Figure 1. Relative abundances of partial (approximately 260 bp) sequences of bacterial 16S rRNA gene were estimated by classification at the phylum level, using MOTHUR with a modified 16S rRNA database from the Ribosomal Database Project. The diverse phylum of Proteobacteria is represented at the class level with different shades of green. A vertical line separates the ballast water samples (CB labels) from those of local Norfolk waters (ODUSP and YC). Key for symbols beneath sample labels: Black triangles=local Norfolk water samples; Squares=tanks sampled in June; Dots=tanks sampled in August/September; Black, gray, or red fill=tanks that underwent a 300% (or greater) volume exchange, a 100% volume exchange, or no exchange, respectively. Un. Proteo=unclassified Proteobacteria. 191x113mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2. Cluster diagram for the 19 samples (3 replicates/sample, except for CB36, with only 2 replicates) constructed from a Bray-Curtis similarity matrix of square-root transformed OTU abundances. One thousand bootstrap analyses were conducted. Sample designations: Black triangles=local Norfolk water samples; Squares=tanks sampled in June; Dots=tanks sampled in August/September; Black, gray, or red font and fill=tanks that underwent a 300% (or greater) volume exchange, a 100% volume exchange, or no exchange, respectively. Country (and for USA samples, state) of last port of call indicated: B (Belgium); CDN (Canada); D (Germany); FRA (France); I (Italy); IRL (Ireland); NET (Netherlands); USA-MA (Massachusetts); USA-AL (Alabama). Local water samples indicated as USA-VA (Virginia). 302x498mm (120 x 120 DPI)



Figure 3. Non-metric multidimensional (NMDS) scaling plots in two dimensions constructed from a BrayCurtis matrix of square-root transformed OTU abundances, including environmental variables (ballast water age, salinity, and temperature). Sample designations: Black triangles=local Norfolk water samples; Squares=tanks sampled in June; Dots=tanks sampled August/September; Black, gray, or red fill=tanks that underwent a 300% volume exchange, a 100% volume exchange, or no exchange, respectively. 201x117mm (300 x 300 DPI)


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TOC graphic 338x190mm (96 x 96 DPI)


Bacterial Diversity in Ships' Ballast Water, Ballast-Water Exchange

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