Zooplankton diversity analysis through single-gene sequencing of a community sample
© Machida et al; licensee BioMed Central Ltd. 2009
Received: 6 August 2008
Accepted: 17 September 2009
Published: 17 September 2009
Oceans cover more than 70% of the earth's surface and are critical for the homeostasis of the environment. Among the components of the ocean ecosystem, zooplankton play vital roles in energy and matter transfer through the system. Despite their importance, understanding of zooplankton biodiversity is limited because of their fragile nature, small body size, and the large number of species from various taxonomic phyla. Here we present the results of single-gene zooplankton community analysis using a method that determines a large number of mitochondrial COI gene sequences from a bulk zooplankton sample. This approach will enable us to estimate the species richness of almost the entire zooplankton community.
A sample was collected from a depth of 721 m to the surface in the western equatorial Pacific off Pohnpei Island, Micronesia, with a plankton net equipped with a 2-m2 mouth opening. A total of 1,336 mitochondrial COI gene sequences were determined from the cDNA library made from the sample. From the determined sequences, the occurrence of 189 species of zooplankton was estimated. BLASTN search results showed high degrees of similarity (>98%) between the query and database for 10 species, including holozooplankton and merozooplankton.
In conjunction with the Census of Marine Zooplankton and Barcode of Life projects, single-gene zooplankton community analysis will be a powerful tool for estimating the species richness of zooplankton communities.
The fauna of the world's oceans is dominated in terms of abundance and biomass by drifting organisms collectively referred to as plankton. Plankton occur in all marine waters, throughout all depths, and, for many species, across widespread biogeographical regions. Zooplankton (planktonic animals) support many major fisheries and mediate fluxes of nutrients and chemical elements essential to life on earth. Despite more than a century of sampling the oceans, a comprehensive understanding of zooplankton biodiversity has eluded oceanographers because of the fragile nature and small body size of these organisms, as well as the large number of species from various taxonomic phyla [1, 2]. For many zooplankton groups, there are longstanding and unresolved questions of species identification, systematic relationships, genetic diversity, and biogeography. In light of this, we are working toward a taxonomically comprehensive assessment of zooplankton biodiversity throughout the world's oceans through the international project Census of Marine Zooplankton .
Results and Discussion
BLASTN search results for sequences that showed more than 98% similarity to subject sequences
Figure 2 shows an unrooted neighbour-joining tree of the 1,336 zooplankton COI sequences. Overall, each taxonomic group formed a single cluster including Gastropoda, Chaetognatha, Euphausiacea, Decapoda, Vertebrata, Copepoda, and Cephalopoda. There were also two cases in which the taxonomic assignment did not work well. The first was the occurrence of Hexapoda in various clusters, which rarely occurs in the ocean environment, except pleustonic insects of the genus Halobates. The second was the difficulty of assignment of taxonomic groups due to low BLAST scores and similarities (coloured grey in Figure 2). The most plausible reason for these ambiguities is the paucity of mitochondrial COI sequences for some taxa in the DNA database. In general, the mitochondrial COI gene sequences in the DNA database are biased among taxa, and this bias was assumed to be the main reason for the occurrence of Hexapoda in our analysis. The most efficient solution for these problems will be the expansion of zooplankton DNA barcode, and it is hoped that the progress of the Barcode of Life project  in collaboration with the Census of Marine Zooplankton will fill these gaps.
Comparison of species that occurred in the present study and the SOND cruise
Present Study (%)
SOND Cruise (%)
Furthermore, about 76 vertically stratified zooplankton samples that were collected above 1,000 m were combined to estimate the occurrence of species . In contrast, the present study was conducted based on a single sample collected from a depth of 721 m to the surface. These sampling effort differences may have accounted for the differences in species richness between the SOND cruise and the present study. In addition, the lower species richness in the present study may have been due to our experimental design. In the present study, after construction of the cDNA library from mRNA, the mitochondrial COI genes were amplified with "universal (LCO1490 )" and polyT primers. It is possible that some of the mitochondrial COI gene sequences may not have been amplified due to primer mismatch for some species. Although the single-gene zooplankton community analysis approach is an efficient means of collecting sequence information, given technical difficulties due to primer mismatch, further studies and the development of novel methodologies are required to gain a complete understanding of zooplankton diversity.
Although the estimation of species richness and composition of the community are among the most important aspects of single-gene zooplankton community analysis, these sequence data will be further utilised by construction of a dedicated database. We expect that the accumulation of additional marine animal mitochondrial COI gene sequence data in the barcode project will aid in further clarifying sequences from unknown species. Furthermore, this process of sequence assignment to particular species through database analysis indicated the occurrence of these species in the sampling site for the present study. We have now constructed a publicly accessible zooplankton community analysis database that can be searched using BLASTN .
With regard to the future of zooplankton community genetic analysis, adoption of next-generation sequencing technology should enable researchers to read libraries sufficiently to estimate species richness without extrapolation [23, 24]. We are currently expanding our sampling effort to all oceans to further understand zooplankton biodiversity.
The sample was collected off Pohnpei Island, Micronesia (6°16'N, 162°09'E). Collection was performed with a plankton net (ORI net ) with a 2-m2 mouth opening and 0.69-mm mesh aperture. After removal of large animals (more than about 4 cm at their largest measurement), the sample was split into two fractions: one was preserved in ethanol for barcode analysis and the other was homogenised with TRIZol (Invitrogen) and kept at -80°C. A total wet volume of about 30 mL zooplankton was collected and homogenised with 270 mL TRIZol in this step.
Total RNA extraction and mRNA purification
In the laboratory, total RNA was extracted from the sample following the TRIZol protocol, followed by mRNA purification using Poly(A)Purist MAG (Ambion). A total of 9.6 mL total RNA (aqueous phase) was further purified for mRNA in this step.
Mitochondrial COI gene library construction and sequence analysis
The purified mRNA was used as the template for Creator SMART cDNA Library Construction Kit (BD Biosciences). Using this constructed cDNA library, we amplified mitochondrial COI genes using COI universal (LCO1490)  and polyT primers with restriction sites that were further used to construct a mitochondrial COI gene library with the same kit. We then randomly analysed colonies obtained on agar plates.
BLASTN search and taxonomic assignment
The lengths of all obtained sequences were adjusted to 500 base pairs, and a BLASTN  search against the NCBI non-redundant dataset with default settings was performed with all sequences as queries. Those sequences that did not show any similarity to the mitochondrial COI gene sequences were removed (the search was performed in November 2006). BLASTN search against the NCBI non-redundant dataset was also used to infer species or higher taxonomic groups of mitochondrial COI gene sequences determined in the present study. In the BLASTN result list, the species with the highest score was assigned to each sequence with the following criteria. If the BLASTN score was 100 or more and BLASTN similarity was 98% or more, the name of the resulted species was assigned to the sequence and listed in table 1. If the BLASTN score was 100 or more and BLASTN similarity was 83-98%, the name of higher taxon group to which the resulted species belongs was assigned to the sequence and is shown in the figure 2. If BLASTN scores and similarity values did not reach these values of criteria, 'unknown' was assigned to the sequences and are colored gray in the figure 2.
Removal of PCR recombination, mismatch distribution analysis, rarefaction curve analysis, phylogenetic analysis
To remove sequences produced by PCR recombination, we manually applied a partial treeing approach  to the aligned dataset; although some programs and servers are available for related analysis, none worked appropriately for our analysis. Briefly, after the sequence alignment was adjusted using ClustalX , square distance matrixes of both the left 100 and right 100 base pairs of the aligned sequence were constructed in MEGA3.1 . Then total absolute deviations of each sequence in these matrixes were calculated. As a result, we deleted one sequence that showed a very large deviation from the others. We assumed this was not the only chimera sequence that occurred in the analysis, but it was not possible to eliminate all PCR recombination sequences because of ambiguity. After removing the PCR recombination sequences from the analysis, we again adjusted alignment of the remaining 1,336 sequences using ClustalX. An unrooted phylogenetic tree was constructed using the neighbour-joining method with nucleotide p-distances (alignment gaps were completely deleted) implemented in PAUP*4.10b . The reliability of each tree node was assessed using the bootstrap method with 1,000 replicates. The mismatch distribution was estimated from the distance matrix. The distance matrix was also calculated using PHYLIP3.66 , and the matrix was further used for rarefaction curve and Chao1 calculation using DOTUR .
We are grateful to the captains and crew members of the R. V. Hakuho Maru for their cooperation at sea. We gratefully acknowledge the support of the Alfred P. Sloan Foundation. Additional support for this project was provided to R.J.M. by a Grant-in-Aid for Scientific Research (No. 20241003) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Data Integration & Analysis System from the Ministry of Education, Culture, Sports, Science and Technology of Japan provided funding to R.J.M. and S.N. This study is a contribution from the Census of Marine Zooplankton, an ocean realm field project of the Census of Marine Life (CMarZ).
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