Dataset Information

Metagenomic Sequencing Identifies Highly Diverse Assemblages of Dinoflagellate Cysts in Sediments from Ships' Ballast Tanks.

ABSTRACT: Ships' ballast tanks have long been known as vectors for the introduction of organisms. We applied next-generation sequencing to detect dinoflagellates (mainly as cysts) in 32 ballast tank sediments collected during 2001-2003 from ships entering the Great Lakes or Chesapeake Bay and subsequently archived. Seventy-three dinoflagellates were fully identified to species level by this metagenomic approach and single-cell polymerase chain reaction (PCR)-based sequencing, including 19 toxic species, 36 harmful algal bloom (HAB) forming species, 22 previously unreported as producing cysts, and 55 reported from ballast tank sediments for the first time (including 13 freshwater species), plus 545 operational taxonomic units (OTUs) not fully identified due to a lack of reference sequences, indicating tank sediments are repositories of many previously undocumented taxa. Analyses indicated great heterogeneity of species composition among samples from different sources. Light and scanning electron microscopy and single-cell PCR sequencing supported and confirmed results of the metagenomic approach. This study increases the number of fully identified dinoflagellate species from ballast tank sediments to 142 (> 50% increase). From the perspective of ballast water management, the high diversity and spatiotemporal heterogeneity of dinoflagellates in ballast tanks argues for continuing research and stringent adherence to procedures intended to prevent unintended introduction of non-indigenous toxic and HAB-forming species.

SUBMITTER: Shang L

PROVIDER: S-EPMC6724030 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Metagenomic Sequencing Identifies Highly Diverse Assemblages of Dinoflagellate Cysts in Sediments from Ships' Ballast Tanks.

Shang Lixia L Hu Zhangxi Z Deng Yunyan Y Liu Yuyang Y Zhai Xinyu X Chai Zhaoyang Z Liu Xiaohan X Zhan Zifeng Z Dobbs Fred C FC Tang Ying Zhong YZ

Microorganisms 20190809 8

Ships' ballast tanks have long been known as vectors for the introduction of organisms. We applied next-generation sequencing to detect dinoflagellates (mainly as cysts) in 32 ballast tank sediments collected during 2001-2003 from ships entering the Great Lakes or Chesapeake Bay and subsequently archived. Seventy-three dinoflagellates were fully identified to species level by this metagenomic approach and single-cell polymerase chain reaction (PCR)-based sequencing, including 19 toxic species, 3 ...[more]

PMID: 31405065

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Project description:Global trade by merchant ships is a leading mechanism for the unintentional transfer of marine organisms, including non-indigenous species, to bays and estuaries worldwide. To reduce the likelihood of new invasions, ships are increasingly being required to manage their ballast water (BW) prior to discharge in coastal waters. In the United States, most overseas arrivals have been required to manage BW discharge since 2004, primarily through ballast water exchange (BWE), which flushes out ballast tanks in the open ocean (>200 miles from shore). Studies have found BWE to generally reduce the abundance of organisms, and the amount of water exchanged has been estimated at 96-100%. Despite its widespread use, the overall effect of this management strategy on net propagule supply through time has not been explored. Here, temporal changes in zooplankton concentrations and the volume of BW discharged in Chesapeake Bay, U.S. were evaluated, comparing pre-management era and post-management era time periods. Chesapeake Bay is a large port system that receives extensive BW discharge, especially from bulk cargo vessels (bulkers) that export coal overseas. For bulkers arriving from overseas, mean zooplankton concentrations of total and coastal indicator taxa in BW did not decline between pre- (1993-2000) and post management (2012-2013) eras, when controlling for season and sampling method. Moreover, bulkers discharged 21 million tonnes (82% of total for Chesapeake Bay) of overseas BW in 2013, representing a 374% increase in volume when compared to 2005. The combination of BW discharge volume and zooplankton concentration data indicates that (a) net propagule supply by bulkers has increased since BWE began in Chesapeake Bay; and (b) changes in vessel behaviour and trade have contributed strongly to this outcome. Specifically, the coal-driven increase in BW discharge volume from 2005-2013, concurrent with the onset of BWE regulations, worked to counteract intended results from BW management. A long-term analysis of bulker arrivals (1994-2013) reveals a 20-year minimum in arrival numbers in 2000, just when the implementation of BWE began. This study underscores the need to consider shifts in trade patterns, in order to advance and evaluate effective management strategies for biological invasions.

Project description:Over the past several decades, much attention has been focused on the dispersal of aquatic nonindigenous species via ballast tanks of shipping vessels worldwide. The recently reclassified dinoflagellate Pseudocochlodinium profundisulcus (previously identified as Cochlodinium sp., Cochlodinium geminatum, or Polykrikos geminatus) was not reported in China until 2006. However, algal blooming events caused by this organism have been reported almost every year since then in the Pearl River Estuary and its adjacent areas in China. Whether P. profundisulcus is an indigenous or an invasive species has thus become an ecological question of great scientific and practical significance. In this study, we collected the sediments from ballast tanks of ships arriving in the ports of China and North America and characterized dinoflagellate resting cysts via a combined approach. We germinated two dark brownish cysts from the tank of an international ship (Vessel A) arriving at the Jiangyin Port (China) into vegetative cells and identified them as P. profundisulcus by light and scanning electron microscopy and phylogenetic analyses for partial LSU rDNA sequences. We also identified P. profundisulcus cyst from the ballast tank sediment of a ship (Vessel B) arriving in the port of North America via single-cyst PCR and cloning sequencing, which indicated that this species could be transported as resting cyst via ship. Since phylogenetic analyses based on partial LSU rDNA sequences could not differentiate all sequences among our cysts from those deposited in the NCBI database into sub-groups, all populations from China, Australia, Japan, and the original sources from which the cysts in the two vessels arrived in China and North America were carried over appeared to share a very recent common ancestor, and the species may have experienced a worldwide expansion recently. These results indicate that P. profundisulcus cysts may have been extensively transferred to many regions of the world via ships' ballast tank sediments. While our work provides an exemplary case for both the feasibility and complexity (in tracking the source) of the bio-invasion risk via the transport of live resting cysts by ship's ballast tanks, it also points out an orientation for future investigation.

Project description:BackgroundDinoflagellate cysts (i.e., dinocysts) are biologically and ecologically important as they can help dinoflagellate species survive harsh environments, facilitate their dispersal and serve as seeds for harmful algal blooms. In addition, dinocysts derived from some species can produce more toxins than vegetative forms, largely affecting species through their food webs and even human health. Consequently, accurate identification of dinocysts represents the first crucial step in many ecological studies. As dinocysts have limited or even no available taxonomic keys, molecular methods have become the first priority for dinocyst identification. However, molecular identification of dinocysts, particularly when using single cells, poses technical challenges. The most serious is the low success rate of PCR, especially for heterotrophic species.MethodsIn this study, we aim to improve the success rate of single dinocyst identification for the chosen dinocyst species (Gonyaulax spinifera, Polykrikos kofoidii, Lingulodinium polyedrum, Pyrophacus steinii, Protoperidinium leonis and Protoperidinium oblongum) distributed in the South China Sea. We worked on two major technical issues: cleaning possible PCR inhibitors attached on the cyst surface and designing new dinoflagellate-specific PCR primers to improve the success of PCR amplification.ResultsFor the cleaning of single dinocysts separated from marine sediments, we used ultrasonic wave-based cleaning and optimized cleaning parameters. Our results showed that the optimized ultrasonic wave-based cleaning method largely improved the identification success rate and accuracy of both molecular and morphological identifications. For the molecular identification with the newly designed dinoflagellate-specific primers (18S634F-18S634R), the success ratio was as high as 86.7% for single dinocysts across multiple taxa when using the optimized ultrasonic wave-based cleaning method, and much higher than that (16.7%) based on traditional micropipette-based cleaning.DiscussionThe technically simple but robust method improved on in this study is expected to serve as a powerful tool in deep understanding of population dynamics of dinocysts and the causes and consequences of potential negative effects caused by dinocysts.

Project description:BackgroundDinoflagellates are a ubiquitous and ecologically important component of marine phytoplankton communities, with particularly notable species including those associated with harmful algal blooms (HABs) and those that bioluminesce. High-throughput sequencing offers a novel approach compared to traditional microscopy for determining species assemblages and distributions of dinoflagellates, which are poorly known especially in Australian waters.ResultsWe assessed the composition of dinoflagellate assemblages in two Australian locations: coastal temperate Port Phillip Bay and offshore tropical waters of Davies Reef (Great Barrier Reef). These locations differ in certain environmental parameters reflecting latitude as well as possible anthropogenic influences. Molecular taxonomic assessment revealed more species than traditional microscopy, and it showed statistically significant differences in dinoflagellate assemblages between locations. Bioluminescent species and known associates of HABs were present at both sites. Dinoflagellates in both areas were mainly represented by the order Gymnodiniales (66%-82% of total sequence reads). In the warm waters of Davies Reef, Gymnodiniales were equally represented by the two superclades, Gymnodiniales sensu stricto (33%) and Gyrodinium (34%). In contrast, in cooler waters of Port Phillip Bay, Gymnodiniales was mainly represented by Gyrodinium (82%). In both locations, bioluminescent dinoflagellates represented up to 0.24% of the total sequence reads, with Protoperidinium the most abundant genus. HAB-related species, mainly represented by Gyrodinium, were more abundant in Port Phillip Bay (up to 47%) than at Davies Reef (28%), potentially reflecting anthropogenic influence from highly populated and industrial areas surrounding the bay. The entire assemblage of dinoflagellates, as well as the subsets of HAB and bioluminescent species, were strongly correlated with water quality parameters (R2 = 0.56-0.92). Significant predictors differed between the subsets: HAB assemblages were explained by salinity, temperature, dissolved oxygen, and total dissolved solids; whereas, bioluminescent assemblages were explained only by salinity and dissolved oxygen, and had greater variability.ConclusionHigh-throughput sequencing and genotyping revealed greater diversity of dinoflagellate assemblages than previously known in both subtropical and temperate Australian waters. Significant correlations of assemblage structure with environmental variables suggest the potential for explaining the distribution and composition of both HAB species and bioluminescent species.

Dataset Information

Metagenomic Sequencing Identifies Highly Diverse Assemblages of Dinoflagellate Cysts in Sediments from Ships' Ballast Tanks.

Publications

Metagenomic Sequencing Identifies Highly Diverse Assemblages of Dinoflagellate Cysts in Sediments from Ships' Ballast Tanks.

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