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Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
1 Oregon State University, Corvallis, OR.
Toxic dinoflagellates of the genus Alexandrium are the primary organisms responsible for seasonal harmful algal blooms (HABs) in coastal New England waters. Members of the genus Alexandrium can produce powerful neurotoxins that accumulate in filter-feeding shellfish, causing debilitating or lethal paralytic shellfish poisoning (PSP) in humans and other higher trophic consumers (1).
To better understand the formation and persistence of HABs, it is important to have accurate measures of population dynamics, specifically growth rate and abundance of toxic cells. Recently, molecular assays have been used to quantify the abundance of the HAB organism Pfiesteria piscicida (2), and similar assays may be useful for efficient analysis of field samples and for prediction of potential A. fundyense HABs. Analogous molecular tools could be used to assess growth rate of A. fundyense populations.
This project employed gene sequencing and specific primer design targeting two Alexandrium fundyense (CA28) gene fragments: one encoding for ribulose-1,5-bisphosphate carboxylase (RubisCO, rbcL); the other encoding for large subunit ribosomal RNA (LSU). This research also explored the use of quantitative polymerase chain reaction (QPCR) to assess growth rate through rbcL gene expression and to quantify cell density through LSU gene abundance. QPCR is highly sensitive, enabling users to monitor increases in PCR product formation during amplification (3). By using an appropriate standard, the starting quantity of the specific mRNA or DNA target in a sample can be calculated.
The rbcL gene was chosen because dinoflagellates, unlike other photosynthetic eukaryotes, express only a Form II RubisCO (4). RubisCO is the key enzyme for fixing CO2 into organic cellular components during the Calvin-Benson cycle. Measurement of rbcL gene expression may be a useful marker for determining changes in cellular growth rate. The LSU gene has a high copy number and is species-specific, making it a good candidate for quantifying CA28 cell density through QPCR.
Degenerate rbcL primers, based on three published dinoflagellate rbcL sequences (GenBank as of June 2002) were used to PCR amplify a weak 320 base pair product (5). This product was sequenced, and the resulting sequences were aligned and used to design specific rbcL primers (forward primer 5'-CACTTTGCAGCTGAGTCTTCCA-3', reverse primer 5'-CAACGCATCCACGGTTTTTGTG-3') that target an 81 base pair fragment of the rbcL gene.
The LSU forward primer (5'-GGAATGCAAAGTGGGTGG-3') was designed from published Alexandrium strain sequences (6). A previously designed oligonucleotide (NA1) was used as a reverse primer. The NA1 oligonucleotide is specific to toxic North American ribotypes of the Alexandrium fundyense/tamarense/catenella species complex (7). These primers targeted a 174 base pair sequence.
Gradient PCR with an annealing temperature range of 50–70 °C was used to determine the highest annealing temperature possible for optimal product formation and primer specificity. Optimal annealing temperatures for the specific rbcL and LSU primers were 63.5 °C and 58 °C respectively.
The specific rbcL primers produced robust amplification of the rbcL gene fragment as compared to the degenerate primers (Fig.1A). The LSU primer pair also produced strong amplification and was highly specific to Alexandrium when tested against members of three other dinoflagellate genera, Gymnodinium sp. (CCMP1937), Lingulodinium polyedra (GPES22), and Amphidinium carterae (Amphi) (Fig. 1B).
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The specific rbcL primers designed and tested during this project may be used for further research on rbcL expression to determine if there is a correlation between growth rate and rbcL message abundance. This could be a valuable tool for rapid analysis of Alexandrium growth rate in field populations.
The LSU primers described here may be useful for analysis of Alexandrium cell densities within mixed field samples, augmenting our abilities to monitor Alexandrium fundyense population density in the field. Future work is needed to optimize conditions for DNA extraction from mixed samples containing known A. fundyense cell densities. If successful, A. fundyense cell numbers in field samples could be quantified by QPCR determination of LSU abundance through comparison to a standard curve.
This work was supported by NSF-REU site grant (OCE-0097498), Boston University Marine Program and Woods Hole Oceanographic Institution.
Literature Cited
This article has been cited by other articles:
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  L. Galluzzi, A. Penna, E. Bertozzini, M. Vila, E. Garces, and M. Magnani Development of a Real-Time PCR Assay for Rapid Detection and Quantification of Alexandrium minutum (a Dinoflagellate) Appl. Envir. Microbiol., February 1, 2004; 70(2): 1199 - 1206. [Abstract] [Full Text] [PDF]
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 M. Galac, D. Erdner, D. M. Anderson, and S. Dyhrman Molecular Quantification of Toxic Alexandrium fundyense in the Gulf of Maine Biol. Bull., October 1, 2003; 205(2): 231 - 232. [Full Text] [PDF]
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