All aboard! A biological survey of ballast water onboard vessels spanning the North Atlantic Ocean

Marine Pollution Bulletin 87 (2014) 201–210

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

All aboard! A biological survey of ballast water onboard vessels spanning the North Atlantic Ocean Jamie L. Steichen a,⇑, Anja Schulze a,c, Robin Brinkmeyer a,b, Antonietta Quigg a,c a

Department of Oceanography, Texas A&M University, 3146 TAMU, College Station, TX 77843, United States Department of Marine Science, Texas A&M University at Galveston, 200 Seawolf Parkway, Galveston, TX 77553, United States c Department of Marine Biology, Texas A&M University at Galveston, 200 Seawolf Parkway, Galveston, TX 77553, United States b

a r t i c l e

i n f o

Article history: Available online 28 August 2014 Keywords: Ballast water Dinoflagellates Diatoms Galveston Bay Invasive species

a b s t r a c t Global movement of nonindigenous species, within ballast water tanks across natural barriers, threatens coastal and estuarine ecosystem biodiversity. In 2012, the Port of Houston ranked 10th largest in the world and 2nd in the US (waterborne tonnage). Ballast water was collected from 13 vessels to genetically examine the eukaryotic microorganism diversity being discharged into the Port of Houston, Texas (USA). Vessels took ballast water onboard in North Atlantic Ocean between the Port of Malabo, Africa and Port of New Orleans, Louisiana, (USA). Twenty genera of Protists, Fungi and Animalia were identified from at least 10 phyla. Dinoflagellates were the most diverse and dominant identified (Alexandrium, Exuviaella, Gyrodinium, Heterocapsa, Karlodinium, Pfiesteria and Scrippsiella). We are reporting the first detection of Picobiliphytes, Apusozoa (Amastigomonas) and Sarcinomyces within ballast water. This study supports that global commerce by shipping contributes to long-distance transportation of eukaryotic microorganisms, increasing propagule pressure and invasion supply on ecosystems. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Over the last century, propagule pressure of non-indigenous (NIS) species has increased, especially in estuaries and coastal waters. Ballast water (BW) is thought to be the primary vector in dispersal and introduction of NIS species of aquatic organisms to ports around the world (Minton et al., 2005; Verling et al., 2005) and new invasion pathways are developing as a result of increasing trade and expanding shipping transport routes (Wonham, 2006). Shipping not only has the ability to increase the frequency but also the volume of these introductions (Lodge, 1993). BW transport from port to port has been attributed to the movement of organisms from their native habitat across natural barriers to new environments (Carlton and Geller, 1993; Drake et al., 2002; Hallegraeff, 1993; Smayda, 2002). After multiple introductions, a species is more likely to become an established NIS or even an invasive species (Wonham, 2006; Carlton, 1985). Zebra and quagga mussels and Chinese mitten crab have already become successful invaders as a result of BW discharge (Benson et al., 2012; Richerson, 2013; Cohen and Carlton, 1997). Although biodiversity may not increase or decrease in the area of interest the species composition may change from native to non-native biota. Maintaining the native ⇑ Corresponding author. Tel.: +1 409 740 4990; fax: +1 409 740 5001. E-mail address: [email protected] (J.L. Steichen). http://dx.doi.org/10.1016/j.marpolbul.2014.07.058 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.

biodiversity within natural systems is important to the resilience and the stability of the communities (Holling, 1973). BW is taken onboard vessels to maintain vessel stability while in transit and then must be managed (i.e. exchanged) before being discharged into other coastal locations. BW exchange is conducted in overseas waters (>200 nm from any shoreline) to replace the biologically diverse coastal water in the BW tanks that may have been taken onboard in the port of origin. For the empty and refill method of BW exchange to be effective, 100% of the BW must be emptied in the open ocean before the tank can be refilled (USCG, 2012). For an efficient exchange utilizing the flow-through method, open ocean water equaling three times the volume of the ballast tank capacity must be pumped through the BW tank. When these management methods are conducted properly, 99% of the initial coastal water should be replaced and over 90% of the coastal zooplankton can be removed from the ballast tanks (Minton et al., 2005; Ruiz et al., 2000). However, there is an exception to the rule for management of BW. Vessels sailing within 200 nm from shore do not have to conduct BW exchange and can discharge their BW ‘coastwise’ directly into port. With increased global distribution of phytoplankton via BW, the communities within port ecosystems have the potential to become altered and biotically homogenized (Drake and Lodge, 2004; Rahel, 2002). Invasive species of phytoplankton must undergo a threestep process before they can successfully invade a habitat

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including: a regional translocation (i.e. BW transport), colonization (i.e. algal bloom) and achievement of competitive dominance (i.e. native biota displaced by invasive species) (Smayda, 2002). For example, the movement and subsequent invasion of the dinoflagellate Prorocentrum minimum to new regions within the Baltic Sea is has been linked to BW dispersal (Olenina et al., 2010). This invasion of P. minimum was shown to displace the native phytoplankton community within the Baltic Sea (Olenina et al., 2010). P. minimum, as many other types of phytoplankton, is capable of tolerating a wide range of salinities as well as producing viable cysts after spending time (>3 months) in unfavorable conditions such as those in a ballast tank (Grzebyk et al., 1997). Dinoflagellates can enter a resting or dormant cyst stage in unfavorable conditions, and/or can switch feeding modes (autotrophic M mixotrophic M heterotrophic) increasing their ability to survive long journeys within ballast tanks (Hallegraeff, 1998; Hallegraeff and Bolch, 1992). Cysts allow dinoflagellates to withstand environmental changes, remaining viable for up to 10 years or more until growth conditions are favorable again (Ribeiro et al., 2011). Not all diatoms and dinoflagellates in the vegetative life stage are capable of tolerating the same variations in environmental conditions that they can while in the cyst stage. The generation time of phytoplankton typically will last from hours to a few days. If the phytoplankton enters into the cyst life stage, they can remain viable for upwards of 100 years (Ribeiro et al., 2011). Galveston Bay is the largest estuary in Texas (Gulf of Mexico, USA), and is highly productive in terms of oyster and seafood production (brown and white shrimp, blue crabs and oysters) second only to Chesapeake Bay in the US (Martin et al., 1996; Lester and Gonzalez, 2011). Studies in this ecosystem are important given the frequency and magnitude of ship traffic and BW discharge into its three ports (Galveston, Houston and Texas City). Steichen et al. (2012) reported that more than 45,000 vessels traveled across Galveston Bay between 2005 and 2010, discharging a total of 1.2  108 metric tons of BW into the Bay itself. BW discharge was found to be an important propagule source of dinoflagellates based on the origin of vessels arriving to Galveston Bay from both domestic and foreign ports of origin (Steichen et al., 2012). Galveston Bay receives more BW discharge than both Chesapeake and San Francisco Bays combined (Steichen et al., 2012; Steichen, 2013). This is important considering the Chesapeake and San Francisco bays are two highly invaded estuarine systems (Cohen and Carlton, 1995, 1998). The goal of this study was to identify the eukaryotic diversity (18S rDNA community primer) including diatoms and dinoflagellates (18s rDNA specific primers), within BW tanks of vessels entering Galveston Bay. To accomplish this, we examined BW from vessels crossing the northern Atlantic Ocean from the Port of Malabo on the West African coast to the Port of New Orleans, Louisiana, (USA). A highly diverse aquatic eukaryotic community, from diatoms and dinoflagellates, to fungi and copepods, was revealed from within the BWs tanks of vessels entering Galveston Bay.

2. Methods 2.1. Sample collection A shipping agent working at a terminal within the POH collected BW samples from vessels. The shipping agent communicated with the vessel captains and BW samples were given on a voluntary basis. Vessels were sampled at various times between May 2007 and March 2010 (Table 1). Per request, the shipping agent and the identity of the vessels remain anonymous. The captains of provided a BW report regarding the time and location

(latitude, longitude) of where the BW was taken onboard. All ships sampled were general cargo vessels. Ships were labeled S1 through S13 corresponding to the location where BW exchanges occurred prior to entering the POH is shown in Fig. 1. The vessels that were sampled in the POH had traveled westward across the North Atlantic Ocean (Fig. 1; Table 1). Samples were collected in a dark acid-washed container and placed on ice for transport to the laboratory. The BW samples were filtered onto a 0.22 lm Sterivex GP (Millepore) cartridge filter using a Masterflex peristaltic pump and tubing (Cole Parmer Instrument Company, Vernon Hills, IL). The filter was stored at 80 °C until DNA extraction was performed. Salinity of the BW sample was measured using a refractometer; all salinity results will be presented on the unit-less practical salinity scale (Table 1). 2.2. Classification of coastwise or overseas BW BW is managed or exchanged in various locations within the coastal and open ocean environments. The National Ballast Information Clearinghouse (NBIC) has developed two categories to better describe the origin of BW. When BW management is conducted or BW is taken onboard a vessel within the Exclusive Economic Zone (EEZ; <200 nm of any shoreline) the BW is termed ‘‘coastwise’’. Vessels that take on or manage BW beyond the EEZ (>200 nm from a shoreline) the BW is termed ‘‘overseas’’. These two categories are based on the definitions used in BW reports submitted and cataloged by the National Ballast Information Clearinghouse of coastwise and overseas. This criterion was applied to the BW samples in this study (Table 1). 2.3. Genetic analysis 2.3.1. Extraction of nucleic acids Genomic DNA was extracted from filters with a cetyltrimethylammonium-bromide (CTAB; 3%)-chloroform isoamyl-alcohol method modified from Doyle and Doyle (1987). Quality and quantity of DNA was determined spectrophotometrically (Nanodrop1000 spectrophotometer). 2.3.2. PCR amplification of 18S rDNA PCR reactions were performed in 50 lL volumes containing approximately 150 ng of template DNA, 10 PCR reaction buffer with 15 mM MgCl2 (Roche Applied Science, Manheim, Germany), 50 lM of each deoxynucleotide, 0.1% bovine serum albumin 1 U Roche Taq DNA polymerase (Roche Applied Science, Manheim, Germany), 10 lM of each primer, and 0.5 lL dimethyl sulfoxide. PCR cycling was conducted using an Eppendorf Mastercycler gradient thermal cycler. Primer sets were selected based on the proven success in amplifying target DNA from environmental water samples and they can be used to identify a wide range of organisms (Giovannoni et al., 1988; Godhe et al., 2008; Oldach et al., 2000; van Hannen et al., 1998; Wang et al., 2005). The primer sets used were designed to identify organisms to the phyla level and in some cases the quality of the DNA allowed resolution to species level (van Hannen et al., 1998). In addition to targeting the more general aquatic eukaryotic community we wanted to target dinoflagellates and diatoms more specifically. To identify the aquatic eukaryotic community, we used the primer set developed by van Hannen et al. (1998): 1427F (50 TCTGTGATGCCCTTAGATGTTCTGGG-30 ) with a 40-bp GC-rich clamp and 1616R (50 -GCGGTGTGTACAAAGGGCAGGG-30 ). The PCR temperature cycling conditions for the 1427F/1616R primer set were: 1 denaturing step at 94 °C for 5 min followed by 25 cycles of 94 °C for 0.5 min, 52 °C for 1 min and 68 °C for 1.5 min and a final extension step of 68 °C for 10 min (van Hannen et al., 1998).

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Table 1 Information pertaining to ballast water onboard vessels at time of sampling in the Port of Houston. Ship name and location correspond to Fig. 1. Ballast water location coordinates show management site (i.e. where last reported exchange occurred). Salinity of BW is shown in the unitless practical salinity scale. Due to anonymity requested by the vessel captains not all information was provided for each sample. Vessel

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13

BW Location Latitude

Longitude

29°560 04N 25°430 00N 20°010 24N 13°400 54N 14°040 00N 26°120 00N 29°490 24N 01°370 30N 05°550 60N 05°490 60N 02°340 42N 01°300 00N 03°400 00N

090°110 11W 089°520 36W 081°050 36W 077°230 24W 068°510 00W 054°350 24W 050°310 18W 032°390 48W 021°550 60W 021°030 00W 016°030 30W 015°160 00W 009°010 00E

Salinity

Age (d)

Distance (nm)

BW Type

2 36a 35a 35a 38 35 39 36 30 35 36 36 3

8 NA NA NA 49 8 48 16 20 33 29 21 42

0 258 40 214 102 646 744 433 545 496 417 407 4

CW OS CW OS CW OS OS OS OS OS OS OS CW

a Indicates salinity of waters at the location where and when ballast water was taken onboard from http://www.nodc.noaa.gov/. Age indicates time in days the ballast water was held in the tanks before sample collection (NA indicates unknown date BW was taken onboard sampled vessel). Distance from nearest land is shown in nautical miles (nm). BW Type indicates coastwise (CW) or overseas (OS) origin of sample. Coastwise BW was taken onboard <200 nm from shore and overseas BW was taken onboard >200 nm from shore.

Fig. 1. Samples were collected from vessels that took ballast water onboard at these locations before entering the Port of Houston (29°34N, 94°50W). Numbers are in ascending order from the POH eastward to the Port of Malabo, Africa.

The dinoflagellate community DNA was targeted using the PCR primer set of Wang et al. (2005): DinoF (50 -CGAT TGAGTGATCCGGTGAATAA-30 ) with a 40 bp GC-rich clamp, and 4618R (50 -TGATCCTTCTGCAGGTTCACCTAC-30 ) was utilized to amplify the targeted DNA fragment. The temperature cycling conditions for the DinoF/4618R primers were as follows: 1 denaturing step at 94 °C for 5 min followed by 40 cycles of 94 °C 30 s, 55 °C for 30 s, and 72 °C for 40 s, then a final extension step of 72 °C for 5 min (Oldach et al., 2000). DNA was amplified utilizing the primer set 1209f (50 - CAGGTCTGTGATGCCCTT-30 ) (Giovannoni et al., 1988) and Diat18SR1

(5’- CAATGCAGWTTGATGAWCTG-3’) (Godhe et al., 2008). We ran DGGE with fragments of DNA that were isolated utilizing a diatom specific primer set (1209F/Diat18SR1) that had previously been used for qPCR. To utilize this primer set in DGGE, we attached a 40 bp GC- rich clamp to the forward primer (1209f), which was developed by Giovanonni et al. (1988). Godhe et al. (2008) designed the reverse primer for identification of the diatoms to the class level. This diatom primer amplified a DNA fragment of approximately 180 base pairs in length with a majority of this region being conserved across diatom lineages. The temperature cycling for the diatom primers set is: 1 denaturing step at 94 °C

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for 5 min followed by 30 cycles of 94 °C 1 m, 48 °C for 1 m, and 72 °C for 1 m, then a final extension step of 72 °C for 5 min (modified in this study from Godhe et al., 2008). PCR products were visualized on 1.5% agarose gels. 2.3.3. Denaturing gradient gel electrophoresis (DGGE) DGGE was performed according to Muyzer and Smalla (1998) to compare the genetic diversity between BW samples. DGGE was conducted separately for each of the aquatic eukaryote primer set (1427F/1616R) and the dinoflagellate and diatom community primer sets (DinoF/4618R and 1209f/Diat18SR1 respectively). The PCR products were run on a separate 1.5 mm thick vertical gel containing 8% (w/v) polyacrylamide (37.5:1 acrylamide:bisacrylamide) and a linear gradient of the denaturants urea and formamide, increasing from 45% at the top of the gel to 55% at the bottom for the aquatic eukaryotic and diatom primer sets and 53–57% for the dinoflagellate primer set. PCR products (50 lL) along with DGGE loading buffer (12 lL) were loaded into the individual lanes. Electrophoresis was performed in a tank containing 1 TAE buffer and 75 V was applied to the submerged gels for 17 h. Nucleic acids were visualized by staining for 30 min in a SyberGold staining bath, containing SyberGold (25 lL) and 1 TAE (250 mL). Gels were viewed on the Bio-Rad GelDoc XR system. The digitized images were inverted using the Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA). DGGE bands were carefully excised from the polyacrylamide gel and stored in PCR grade water (50 lL) for 12 h at 4 °C to elute DNA. The excised bands were analyzed by a second PCR-DGGE step to ensure that a single band was obtained from each DNA fragment. The final bands were excised and the DNA was eluted at 4 °C for 12 h. This final DNA was then diluted (5–10 depending on concentration of DNA) with sterile distilled water in preparation for sequencing. 2.3.4. DNA sequence and phylogenetic analysis Bands containing DNA fragments were excised from the DGGE gels (Figs. 2 and 3). This double stranded DNA was eluted and then used as templates for nucleotide sequencing following the protocols of the Big Dye Terminator v. 3.1 Sequencing Kit (Applied Biosystems, Foster City, CA). The fragments of DNA amplified from the eukaryotic community, dinoflagellates and diatoms were sequenced on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). We found the closest genetic matches available utilizing the BLAST (Basic Local Alignment Search Tool (Altschul et al., 1997). GenBank sequences, having at least 90% similarity to the unknown sequences in our samples, were included in the phylogenetic analysis. The Apicomplexa, Toxoplasma gondii (a close relative to dinophyta), was the outgroup

Fig. 2. DGGE image showing bands from gel ran with eukaryotic (bold) and diatom (not bold) primer sets. Vessel assignments correspond to the following lanes on the gel as follows: S1:1, S3:2–3, S4:4, S5:5, S7:6–7, S8:8, S11:9–10 and S13:11.

Fig. 3. DGGE image showing bands from gel ran with dinoflagellate primer set. Vessel assignments are labeled at the top of each lane of the gel.

taxon utilized for the dinoflagellate tree (Burkholder et al., 2007). These diatom DNA fragments were shortened for the phylogenetic analysis (to 66 base pairs) to allow for more specific differentiation between the identified genera. The stramenopiles, Bolidomonas pacifica and B. mediterranea share an immediate common ancestor with the diatoms and were used as the outgroup for this tree (Sorhannus, 2007). The sequences downloaded from GenBank and the sequences from the unknown organisms were aligned using CLUSTAL W within the Molecular Evolutionary Genetics Analysis (MEGA) 5.0 program (Tamura et al., 2011) to create their respective phylogenetic trees. Sequences were further aligned manually. Evolutionary distances were constructed using the Maximum Likelihood method (Felsenstein, 1985) with discrete Gamma distribution in addition to the Jukes Cantor model with NeighborJoining algorithm (Jukes and Cantor, 1969; Saitou and Nei, 1987). The bootstrap values (>60 after 5000 re-samplings) from both the Maximum likelihood (MLE) and Neighbor Joining (NJ) methods are shown on each node (MLE/NJ). Sequences of the partial 18S rDNA obtained from this study were not submitted to GenBank, as they are all less than 200 base pairs (Benson et al., 2014). The alignments used to construct the phylogenetic trees are provided as supplementary material.

3. Results 3.1. Aquatic eukaryotes, diatoms and dinoflagellates BW samples were genetically examined to compare and contrast organisms being transported in BW tanks of vessels calling at the POH to assess potential invasions and propagules (Figs. 4– 6). The volume of BW that was filtered from each of these vessels for genetic analysis ranged from 1.5 to 8.5 L for coastwise vessels and 2–11 L for overseas vessels. Thirty percent of the vessels sampled had coastwise BW onboard (S1, S3, S5 and S13) and the remaining seventy percent of the sampled vessels (S2, S4, S6– S12) had BW of overseas origin (Fig. 1; Table 1). When combining the results from all BW samples, we identified 44 species from ten Phyla including: Bacillariophyta, Dinophyta, Chrysophyta, Chlorophyta, Picobiliphyta, Sarcomastigophora, Ascomycota, Apusozoa, Ciliophora and Arthropoda (Figs. 4–6; Table 2). Two genera of Fungi were identified including Fusarium and Sarcinomyces. The copepods identified included members the phyla Calanoida, Copepoda, Podoplea and the genus Apocyclops (Table 2). Dinophyta were the most common phylum of eukaryotes identified among the BW samples; they were present in 9 of the 13 BW samples. All of the dinoflagellate genera identified here have previously been found in BW samples (Burkholder et al., 2007; David et al., 2007; Doblin et al., 2004; Drake et al., 2005; Olenina et al., 2010; Pertola et al., 2006). Chrysophytes were identified in 3 BW

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Fig. 4. Phylogenetic tree showing the aquatic eukaryotic diversity found within ballast water samples S1–S13. Taxonomic label on each branch is followed by ship in which the organism was identified. The evolutionary history was inferred by using the Maximum Parsimony (tree shown) and Neighbor-Joining methods based on the Kimura 2parameter model (Saitou and Nei, 1987; Kimura, 1980). This phylogenetic tree included 137 nucleotide sequences and a total of 151 positions in the final dataset. Cirripedia was used as an outgroup for the tree. The percentage (>50%) of replicate trees in which the associated taxa clustered together in the bootstrap test (5000 replicates) are shown above the branches (NJ/MP; Felsenstein, 1985). Clades were collapsed to lowest common taxonomic level.

Fig. 5. Phylogenetic tree showing the dinoflagellate diversity found within ballast water samples S1–S13. Taxonomic label on each branch is followed by ship in which the organism was identified. The evolutionary history was inferred using both the Maximum Likelihood (tree shown) and Neighbor-Joining methods based on the Kimura 2-parameter model (Saitou and Nei, 1987; Kimura, 1980). The alignment included 74 nucleotide sequences and 70 nucleotide positions. The percentage (>50%) of replicate trees in which the associated taxa clustered together in the bootstrap test (5000 replicates) are shown above the branches (NJ/MLE; Felsenstein, 1985). Clades were collapsed to lowest common taxonomic level. Toxoplasma gondii (an apicomplexa) was used as the outgroup taxon.

Fig. 6. Phylogenetic tree showing the diatom diversity found within ballast water samples S1–S13. Taxonomic label on each branch is followed by ship in which the organism was identified. The evolutionary history was inferred using both the Maximum Likelihood (tree shown) and Neighbor-Joining methods based on the Kimura 2-parameter model (Saitou and Nei, 1987; Kimura, 1980). This tree was constructed using 28 nucleotide sequences with 66 total positions in the final dataset. The percentage (>50%) of replicate trees in which the associated taxa clustered together in the bootstrap test (5000 replicates) are shown above the branches (NJ/MLE; Felsenstein, 1985). Clades were collapsed to lowest common taxonomic level. Two species of Bolidomonas (Stramenopiles) were used as the outgroup taxa for this tree.

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Table 2 Eukaryotic diversity found within the ballast water samples. Ship names (S1–S13) correspond to vessel locations shown in Fig. 1. Taxa identified in each of the ballast water tanks sample indicated in the respective column with an ‘‘x’’. Groups were identified down to the species level when possible. Eukaryotic representation in ballast water samples Kingdom

Phylum

Identification

S1

Protista

Apusozoa Bacillariophyta

Amastigomonas sp. Actinocyclus actinochilus Ditylum brightwellii Nitzschia sp. Stephanopyxis turris Thalassiosirales Chlorella sp. Dunaliella sp. Chrysophytes Strombidiidae Choanoflagellida Savillea sp. Alexandrium monilatum Exuviella pusilla Gyrodinium sp. Heterocapsa rotundata Karlodinium sp. Pfiesteria piscida Prorocentrum sp. Scrippsiella sp. Picobiliphytes Fusarium sp. Sarcinomyces sp. Ustilaginaceae Apocyclops sp. Calanoida Copepoda Podoplea Eukaryote (JF488799) Uncultured eukaryote (FN690464) Uncultured eukaryote (GU824848) Uncultured eukaryote (GU825493) Uncultured eukaryote (GU825561) Uncultured eukaryote (JQ782380) Uncultured eukaryote (JN090897) Uncultured eukaryote (EF172986) Uncultured freshwater eukaryote (AY919716) Uncultured marine eukaryote (DQ310196) Uncultured marine eukaryote (EF527203) Uncultured marine eukaryote (HM749942) Uncultured marine eukaryote (JF791087) Uncultured marine stramenopile (HQ156894) Uncultured marine stramenopile (JF826323) Uncultured phototrophic eukaryote (DQ222878)

x

Chlorophyta Chrysophyta Ciliophora Choanozoa Dinophyta

Fungi

Picobiliphyta Ascomycota

Animalia

Arthropoda

Uncultured

Total

samples of which 2 were coastwise in origin and 1 overseas (Table 2). Members of Bacillariophyta were identified in two of the vessels sampled (S1 and S13), both with coastwise BW (Fig. 4; Table 2). Three of the bacillariophyta genera we have identified here have been previously found within BW tanks (Burkholder et al., 2007; Klein et al., 2010).

3.2. Coastwise BW Vessels S1, S3, S5 and S13 all contained BW of coastwise origin with salinity ranging from 2 to 38 (Table 1). The age of the BW retrieved from these vessels ranged from 8 to 49 days (Table 1). BW collected from S5 did not contain any identifiable DNA using these three primer sets. However, given that we were able to extract DNA from the S5 sample (3.34 ng L1) and that there were faint bands on the DGGE images (Figs. 2 and 3), an alternative reason maybe that the DNA fragments were not present in a concentration high enough or that their quality was not sufficient for identification of that particular organism.

S2

S3

S4

S5

S6

S7

S8

S9

S10

S11

S12

S13

x x x x x x x x x

x

x x

x x

x x x

x x

x

x x x x

x

x

x x x x

x

x x x

x x

x x

x x

x

x x x x x x x x

x x

x x x x x

x 13

1

9

3

0

8

3

1

1

0

14

0

3

Vessel S1 took on BW in the Port of New Orleans, Louisiana (USA) located on the Mississippi River and contained the highest diversity of all ships sampled with 8 phyla identified including: Dinophyta, Chrysophyta, Picobiliphyta, Sarcomastigophora, Ascomycota, Apusozoa, Ciliophora, and Arthropoda (Fig. 4; Tables 1 and 2). The dinoflagellates identified in this sample include Heterocapsa rotundata and an uncultured dinoflagellate that fell into a sister clade of Pfiesteria (Fig. 5). Two additional phyla of microalgae were identified from S1 including 3 sequences that grouped with Chrysophytes and one with the Picobiliphyte clade. In addition to the microalgae, we identified organisms belonging to 5 other phyla including: Savillea (Choanoflagellate), Fusarium and Sarcinomyces (Fungi), Amastigomonas (Apusozoa), Strombidiidae (Ciliates) and 2 Calanoid copepods (Fig. 4; Table 2). Vessel S3 took on BW 40 nm from shore, showed the third highest diversity of all the samples collected (Tables 1 and 2). Bacillariophyta (Actinocyclus actinochilus and Stephanopyxis turris), Dinophytes (Gyrodinium sp. and Prorocentrum sp.), Chrysophytes and Ascomycetes, more specifically Ustilaginaceae and Copepoda were identified in this BW sample (Fig. 4; Table 2). Vessel S13

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exchanged BW near the Port of Malabo on Bioko Island, Africa (Fig. 1, Table 1). The diatoms identified in this sample include Ditylum brightwellii, Nitzschia sp., and a member of Thalassiorsirales (Fig. 6). 3.3. Overseas BW Vessels S2, S4 and S6–S12 all contained BW of overseas origin with salinities ranging from 30 to 39 (Table 1). The BW had been onboard these vessels ranging from 8 to 48 days. Vessel S12 did not contain any identifiable DNA using these three primer sets. Similar to the outcome of S5, we were able to extract DNA from the S12 sample (8.67 ng L1) and faint bands appeared on the DGGE gels (Figs. 2 and 3) but the concentration may not have been high enough to allow for identification. Dinoflagellates were identified within all of the overseas BW samples (excluding S12) including: S2 and S4 (Gyrodinium sp.), S6 (Pfiesteria and Scrippsiella), S7 (Karlodinium, Gyrodinium and Prorocentrum), S8 (Alexandrium monilatum), S9 and S10 (Prorocentrum) and S11 (Exuviaella pussilla and Gyrodinium). Vessel S4 took on BW approximately 214 nm offshore and in addition to Gyrodinium sp., Chrysophyta, Arthropoda (Copepoda sp.) and an uncultured eukaryote were identified (Figs. 4 and 5; Table 2). The BW from vessel S7 was taken onboard 744 nm from shore and contained a member of a Choanoflagellida, 2 uncultured eukaryotes and 1 uncultured marine stramenopile (Figs. 4 and 5; Table 2). Vessel S11 showed the second highest diversity overall vessels sampled, with 5 identifiable phyla. This vessel contained overseas BW that was taken onboard on the west coast of Africa near the Port of Malabo. This vessel reported having exchanged BW approximately 400 and in addition to the dinoflagellates, we identified 2 species of Chlorophyta (Chlorella and Dunaliella), 1 species of Picobiliphytes, 2 species of Sarcomastigophora (Choanoflagellida and Savillea sp.) and Arthropoda (Apocyclops, Podoplea, and Calanoida were identified (Fig. 4; Table 2). 4. Discussion Galveston Bay receives 1.9  107 metric tons of BW discharge annually (between 2005 and 2010), primarily of coastwise origin (Steichen et al., 2012). Typically voyages that allow vessels to remain within the EEZ are shorter in duration, allowing for increased survival rate among organism entrained in the BW tanks (Lavoie et al., 1999). Therefore, vessels entering and discharging coastwise BW have the higher potential of discharging viable organisms into Galveston Bay. Dinoflagellates have the capability to enter a dormant cyst stage in unfavorable conditions allowing for successful transportation of these organisms in ballast tanks across the otherwise natural borders hence increasing the likelihood of an invasion (Smayda, 2002). Dinoflagellates were identified within 70% of the BW samples analyzed here, some of which are known harmful algal bloom (HAB) producers. While several of the genera of organisms we identified within the BW samples do exist in Galveston Bay, it remains to be determined if the organisms arriving within the BW pose an invasion threat. The identification of the phytoplankton community at the species level is poorly understood within Galveston Bay. Recent studies have been conducted to better understand the phytoplankton community (Quigg et al., 2009; Steichen, 2013) but previously this historical data has been sparse to non-existent. 4.1. Potential invaders from BW Pfiesteria piscicida (HAB species) has been identified previously in Galveston Bay during isolated sa mpling events using genetic

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identification but is not seen in routine sampling and identification using microscopic methods (Quigg et al., 2009). Villareal et al. (2004) identified P. piscicida at several locations within Galveston Bay in 2000–2001. More recently, Steichen (2013) identified P. piscicida in the waters of the POH. Although P. piscicida has been identified on multiple occasions, it has not formed a HAB in Galveston Bay to date. P. piscicida has been given the name of ‘ambush predator dinoflagellate’ due to the lethal toxin it produces to cause massive fish kills (Burkholder and Glasgow, 1997). P. piscicida is a prominent HAB species that has a dormant cyst stage during their life cycle (Burkholder and Glasgow, 1997; Litaker et al., 2002) and has the ability to feed through phagocytosis (Burkholder, 1998). Both of these characteristics could aide in their survival of the inhospitable conditions experienced during a voyage within a BW tank. Given that there are already significant fish kills in this region (>380 million dead fish between 1956 and 2006; Thronson and Quigg, 2008), the repeated introduction of Pfiesteria and other harmful dinoflagellates are of concern to the region. E. pussilla (synonym: Prorocentrum nanum (J. Schiller)), Karlodinium and Scrippsiella identified here in BW samples have not been previously identified within the POH or Galveston Bay, TX (Quigg et al., 2009; Steichen, 2013). These genera have characteristics enabling them to be potentially successful invaders. E. pussilla has a wide geographical range with blooms having been reported in America, Japan, and South Africa (Elbrächter, 1999). Viable cells of Karlodinium and Scrippsiella have previously been collected after a voyage (2–4 days) from a BW tank sample (Burkholder et al., 2007; Hallegraeff and Bolch, 1992). Karlodinium sp. is common in temperate and coastal phytoplankton communities and can produce a toxin (karlotoxin) that deters grazing (Adolf et al., 2007). Karlodinium veneficum has been shown to produce this karlotoxin and successfully out-compete other co-occurring dinoflagellates making it a more successful invader (Hall et al., 2008). Scrippsiella spp. have caused massive fish kills due to oxygen depletion (Hallegraeff, 1993). Scrippsiella spp. have been found previously in BW samples and the recovered resting spores were successfully germinated (Hallegraeff and Bolch, 1992). A. monilatum is a cyst producing dinoflagellate that produces a strong ichthyotoxin causing fish kills (Hallegraeff and Bolch, 1991, 1992). A. monilatum has formed a HAB(s) in Galveston Bay but reportedly there were no fish kills associated with this bloom (Meredith Byrd with Texas Parks and Wildlife Department, pers. comm). Other species of Alexandrium with these similar characteristics have been reported to survive voyages within a BW and have become invasive after being discharged in a new location (Hallegraeff, 1998). Further genetic research is necessary to determine if the bloom forming A. monilatum present was introduced to Galveston Bay via BW. Of the diatoms we identified in BW, species of Actinocyclus, Ditylum, Nitzschia and Thalassiorsirales have previously been found viable in ballast tanks (Burkholder et al., 2007; Klein et al., 2010). We have identified these diatoms within the BW of vessel S13 that had very low salinity and was taken onboard at Port of Malabo, Africa (Fig. 1; Table 1). While these genera have previously been recorded in Galveston Bay, more specific identification is required to better understand the species composition and origin of the diatom community found within Galveston Bay (Quigg et al., 2009; Steichen, 2013). Picobiliphytes are a relatively novel phylum of marine picoplanktonic algae (Not et al., 2007). The organisms in this lineage are thought to be heterotrophic and are presumed to feed on Proteobacteria, Bacteroidetes and large DNA viruses. Picobiliphytes have been genetically identified from a variety of marine systems, including the European coast, the North Atlantic (Not et al., 2007), and the Arctic Ocean (Lovejoy et al., 2006). To our knowledge, this is the first report of picobiliphytes in BW samples. Given the ships

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we examined traversed the northern Atlantic Ocean it seems plausible they may be entrained into the ballast tanks during a midocean BWE due to their known existence in the marine environment. This group of organisms has not previously been identified within Galveston Bay and data is lacking regarding the invasibility potential of this group of organisms. Amastigomonas (synonym: Thecamonas (Larsen and Patterson, 1990) is a cosmopolitan genus of Apusozoa. This heterotrophic marine flagellate feeds on bacteria and has been identified primarily in marine waters. This understudied genus was recently divided this into five new genera by Cavalier-Smith and Chao (2010). These genera include both marine and non-marine species. These flagellated protozoa have not previously been reported in BW and has not been identified from Galveston Bay waters. Two genera of Fungi, Fusarium and Sarcinomyces, were identified in a vessel sample with coastwise BW taken onboard in New Orleans (Tables 1 and 2). Zvyagintsev et al. (2009) reported finding a Fusarium sp. in a BW sample from a vessel that had taken on BW in the Port of Vladivostok (Russia). Species of the genus Fusarium are known to produce mycotoxins, which are harmful to animals (Desjardins and Proctor, 2007). Reports of Sarcinomyces in ballast tanks are nonexistent to our knowledge. Data on fungi within other BW tanks has been lacking or has been characterized as ‘‘other taxa’’ (Gollasch et al., 2000; Zvereva et al., 2012). Due to the toxic nature of Fusarium, future research should be conducted to better understand the fungi community in Galveston Bay waters. 4.2. Evidence of a sufficient BW exchange The lack of identifiable DNA in S5 and S12 may be attributed a number of different factors. These include (i) the age of the BW, (ii) the location where BW was taken on board (i.e. overseas) or (iii) it may indicate that an efficient BWE was conducted and the organisms were adequately flushed out of the BW tanks (Table 1). In the case of S5, a growout experiment was conducted (Steichen, 2013) in which there was no visible growth after 3 weeks with various nutrient concentrations and light exposure. The results of the growout experiment support the finding of low amounts of DNA in this BW sample and may explain why no identifiable organisms were present. 4.3. Risk of invasion via BW This study provides evidence that non-native species are found within BW of vessels entering Galveston Bay en route to the POH. While we did not conduct a quantitative analysis, we can nonetheless report that at least one of each of the organisms we identified was present in its respective ballast tank with their DNA presence. Based on data presented by Steichen et al. (2012), the average volume of BW released from each vessel that discharged BW into Galveston Bay between 2005 and 2010 was 8100 m3 (per discharge event) for vessels of coastwise origin and5100 m3 for vessels originating from overseas. Based on these volumes, we estimate that in the coastwise ballast samples there could be 63300 organisms m3 and 61200 organisms m3 in the BW of overseas origin (Tables 1 and 2) being discharged per vessel.

carrying BW en route to the POH are transporting a wide diversity of organisms some of which may pose a threat to Galveston Bay. Dinoflagellates present a particular concern because they have the capability of surviving in a BW tank for weeks to months. Many harmful and toxin producing dinoflagellates species are capable of producing resting stages (cysts) that can remain viable for long periods (i.e. ship voyage) and then be discharged from the ship at port (Hallegraeff and Bolch, 1992). Vessels arriving from both domestic and foreign ports around the Gulf of Mexico pose the greatest threat for introductions of non-native HAB species into Galveston Bay because of similar climates and geographic proximity (Steichen et al., 2012). We identified seven genera of dinoflagellates that if released into Galveston Bay have the potential of forming a HAB endangering the native biota. The expansion of the Panama Canal is to be completed in 2015. As a result, larger vessels will enter the Gulf of Mexico from Asia with a much shorter voyage time. BW discharge by these vessels is one of the known transport vectors for delivering invasive species to San Francisco Bay, which is considered one of the most heavily invaded estuaries in the world (Cohen and Carlton, 1995, 1998). Hence this increase in ship traffic increases the threat of invasions to Galveston Bay. Further work must be conducted testing the viability of organisms within the ballast tanks of vessels entering Galveston Bay to further assess the risk of invasion via BW to this system. Worldwide commerce, and in turn shipping, will continue to grow and increase in efficiency (i.e. shorter transit times) pushing the effort to continually improve BW management methods. As shown in Steichen (2013) Galveston Bay received over 20  106 mt more discharged BW than both Chesapeake and San Francisco Bays combined from 2008 to 2012. Galveston Bay, along with other coastal regions, is a highly productive ecosystem that must be protected from the increased propagule pressure via BW. Continued monitoring is essential to protect these vulnerable ecosystems as the invasion risk increases due to anthropogenic forcing.

Acknowledgements We would like to thank Dr. Daniel Roelke at Texas A&M University for reading and providing valuable suggestions on previous versions of this manuscript. We also thank the reviewers and editor of Marine Pollution Bulletin that contributed their valuable insight to further improve this work. We extend our gratitude to the various funding agencies of this project including the Advanced Research Program, Texas General Land Office – CMP, the Environmental Protection Agency. This research would not have been possible without the cooperation of the shipping agent in Houston, Texas that collected all the ballast water samples analyzed (whom would like to remain anonymous); We thank you very much for all of your assistance in the collection of these and many other ballast water samples. We extend a special thank you to all of the members (past, present and auxiliary) of the Phytoplankton Dynamics Laboratory at Texas A&M University at Galveston, who have assisted in the collection and processing of these samples.

5. Conclusions This molecular survey found a high diversity of eukaryotic organisms being transported in ballast tanks of vessels entering Galveston Bay. To our knowledge, these findings include the first reporting of Picobiliphytes and Apusozoa identified in BW samples. Further studies are necessary to determine the viability of these organisms after being subjected to BW tank conditions. Vessels

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.marpolbul.201 4.07.058.

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