Microbial Ecology

, Volume 71, Issue 3, pp 530–542 | Cite as

Amplicon-Based Pyrosequencing Reveals High Diversity of Protistan Parasites in Ships’ Ballast Water: Implications for Biogeography and Infectious Diseases

  • K. M. Pagenkopp LohanEmail author
  • R. C. Fleischer
  • K. J. Carney
  • K. K. Holzer
  • G. M. Ruiz
Microbiology of Aquatic Systems


Ships’ ballast water (BW) commonly moves macroorganisms and microorganisms across the world’s oceans and along coasts; however, the majority of these microbial transfers have gone undetected. We applied high-throughput sequencing methods to identify microbial eukaryotes, specifically emphasizing the protistan parasites, in ships’ BW collected from vessels calling to the Chesapeake Bay (Virginia and Maryland, USA) from European and Eastern Canadian ports. We utilized tagged-amplicon 454 pyrosequencing with two general primer sets, amplifying either the V4 or V9 domain of the small subunit (SSU) of the ribosomal RNA (rRNA) gene complex, from total DNA extracted from water samples collected from the ballast tanks of bulk cargo vessels. We detected a diverse group of protistan taxa, with some known to contain important parasites in marine systems, including Apicomplexa (unidentified apicomplexans, unidentified gregarines, Cryptosporidium spp.), Dinophyta (Blastodinium spp., Euduboscquella sp., unidentified syndinids, Karlodinium spp., Syndinium spp.), Perkinsea (Parvilucifera sp.), Opisthokonta (Ichthyosporea sp., Pseudoperkinsidae, unidentified ichthyosporeans), and Stramenopiles (Labyrinthulomycetes). Further characterization of groups with parasitic taxa, consisting of phylogenetic analyses for four taxa (Cryptosporidium spp., Parvilucifera spp., Labyrinthulomycetes, and Ichthyosporea), revealed that sequences were obtained from both known and novel lineages. This study demonstrates that high-throughput sequencing is a viable and sensitive method for detecting parasitic protists when present and transported in the ballast water of ships. These data also underscore the potential importance of human-aided dispersal in the biogeography of these microbes and emerging diseases in the world’s oceans.


Chesapeake Bay Non-indigenous Non-native Parasite Protist Metabarcode Harbor 



The crews of the various cargo ships, the facilities within the Ports of Baltimore and Hampton Roads, and agents T. Parker Host and Capes Shipping Agencies enabled vessel and tank access. Rebecca Dikow assisted with interpreting some phylogenetic analyses and access to computers. Rebecca Dikow, Carly Woltz, and three anonymous reviewers provided useful feedback on the content of this manuscript. Taylor and Joel Callicrate assisted with file formatting. Nancy Rotzel McInerney provided logistical support at CCEG. We thank Roche-454 for providing a GS Junior to CCEG, and assistance with protocols. This project was funded by the Center for Conservation and Evolutionary Genetics and a Smithsonian Institution Next Generation Sequencing Small Grant. Sample collection was supported by funding from the US Coast Guard. Pagenkopp Lohan was funded by a Smithsonian MarineGEO Postdoctoral Fellowship.

Supplementary material

248_2015_684_MOESM1_ESM.pdf (744 kb)
ESM 1 (PDF 744 kb)


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Copyright information

© Springer Science+Business Media New York (outside the USA) 2015

Authors and Affiliations

  • K. M. Pagenkopp Lohan
    • 1
    • 2
    Email author
  • R. C. Fleischer
    • 1
  • K. J. Carney
    • 2
  • K. K. Holzer
    • 2
  • G. M. Ruiz
    • 2
  1. 1.Center for Conservation and Evolutionary Genetics, Smithsonian Conservation Biology InstituteNational Zoological ParkWashingtonUSA
  2. 2.Marine Invasions LaboratorySmithsonian Environmental Research CenterEdgewaterUSA

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