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Molecular detection and species identification of Alexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline.

Jedlicki A, Fernández G, Astorga M, Oyarzún P, Toro JE, Navarro JM, Martínez V - AoB Plants (2012)

Bottom Line: For species-specific determination, the intergenic spacer 1 (ITS1), 5.8S subunit, ITS2 and the hypervariable genomic regions D1-D5 of the large ribosomal subunit of local strains were sequenced and compared with two data sets of other Alexandrium sequences.Our results support the allocation of the Chilean Alexandrium species as a toxic Alexandrium tamarense rather than A. catenella, as currently defined.The results show that real-time PCR is useful to detect Alexandrium intake in filter-feeding molluscs.

View Article: PubMed Central - PubMed

Affiliation: FAVET-INBIOGEN, Faculty of Veterinary Sciences , University of Chile , Santa Rosa No 11.735 (PO 8820808), Santiago , Chile.

ABSTRACT

Background and aims: On the basis of morphological evidence, the species involved in South American Pacific coast harmful algal blooms (HABs) has been traditionally recognized as Alexandrium catenella (Dinophyceae). However, these observations have not been confirmed using evidence based on genomic sequence variability. Our principal objective was to accurately determine the species of Alexandrium involved in local HABs in order to implement a real-time polymerase chain reaction (PCR) assay for its rapid and easy detection on filter-feeding shellfish, such as mussels.

Methodology: For species-specific determination, the intergenic spacer 1 (ITS1), 5.8S subunit, ITS2 and the hypervariable genomic regions D1-D5 of the large ribosomal subunit of local strains were sequenced and compared with two data sets of other Alexandrium sequences. Species-specific primers were used to amplify signature sequences within the genomic DNA of the studied species by conventional and real-time PCR.

Principal results: Phylogenetic analysis determined that the Chilean strain falls into Group I of the tamarensis complex. Our results support the allocation of the Chilean Alexandrium species as a toxic Alexandrium tamarense rather than A. catenella, as currently defined. Once local species were determined to belong to Group I of the tamarensis complex, a highly sensitive and accurate real-time PCR procedure was developed to detect dinoflagellate presence in Mytilus spp. (Bivalvia) samples after being fed (challenged) in vitro with the Chilean Alexandrium strain. The results show that real-time PCR is useful to detect Alexandrium intake in filter-feeding molluscs.

Conclusions: It has been shown that the classification of local Alexandrium using morphological evidence is not very accurate. Molecular methods enabled the HAB dinoflagellate species of the Chilean coast to be assigned as A. tamarense rather than A. catenella. Real-time PCR analysis based on A. tamarense primers allowed the detection of dinoflagellate DNA in Mytilus spp. samples exposed to this alga. Through the specific assignment of dinoflagellate species involved in HABs, more reliable preventive policies can be implemented.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic tree of 80 Alexandrium species andstrains (Atm, A. tamarense; Act, A. catenella;Afu, A. fundyense; Amn, A. minutum; Aaf,A. affine; Atr, A. tropicale; Aty,A. tamiyanavichii; P. micans,Prorocentrum micans) and local Alexandriumspecies Ach01. Sequences were obtained from the GenBank database usingthe keywords ‘Alexandrium LSU rDNA’ or ‘Alexandrium28S’. Phylogenetic trees were generated for an alignment of 81 sequences of641 bp in the D1/D2 region through Bayesian inference in MrBayes V3.2 and maximumlikelihood (ML) in PhyML. Bayesian analysis was carried out with 1 500 000 runs,with five separate initial trees. Convergence was checked through PSRF and AVSF.FigTree V1.3.1 was used as a visual representation of output trees. For MLanalysis these were carried out with 1000 bootstrap replicates.
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PLS033F3: Phylogenetic tree of 80 Alexandrium species andstrains (Atm, A. tamarense; Act, A. catenella;Afu, A. fundyense; Amn, A. minutum; Aaf,A. affine; Atr, A. tropicale; Aty,A. tamiyanavichii; P. micans,Prorocentrum micans) and local Alexandriumspecies Ach01. Sequences were obtained from the GenBank database usingthe keywords ‘Alexandrium LSU rDNA’ or ‘Alexandrium28S’. Phylogenetic trees were generated for an alignment of 81 sequences of641 bp in the D1/D2 region through Bayesian inference in MrBayes V3.2 and maximumlikelihood (ML) in PhyML. Bayesian analysis was carried out with 1 500 000 runs,with five separate initial trees. Convergence was checked through PSRF and AVSF.FigTree V1.3.1 was used as a visual representation of output trees. For MLanalysis these were carried out with 1000 bootstrap replicates.

Mentions: Using 81 sequences of length 641 bp, all aligning in the same D1/D2 LSU rDNA region,from different species of the genus Alexandrium and two strains ofP. micans, the phylogenetic distribution of localAlexandrium strains could be estimated (Fig. 3). Convergence of Bayesian trees was evaluatedthrough AVSF and PSRF. Values were less than 0.01 and ∼1 ±0.005,respectively, suggesting that the distribution reached a stationary phase in both datasets. Additionally, bootstrap values and the logarithm of the likelihood score of theoptimal tree (−4229.43342) extracted by maximum likelihood analysis wereindicative of a precise tree. The results indicate that the localAlexandrium strain is in Group I, dominated by A.tamarense, and is grouped with other previously sequencedAlexandrium strains from Chilean waters (ACC01, ACC02 and ACC07;Fig. 3). Fig. 3


Molecular detection and species identification of Alexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline.

Jedlicki A, Fernández G, Astorga M, Oyarzún P, Toro JE, Navarro JM, Martínez V - AoB Plants (2012)

Phylogenetic tree of 80 Alexandrium species andstrains (Atm, A. tamarense; Act, A. catenella;Afu, A. fundyense; Amn, A. minutum; Aaf,A. affine; Atr, A. tropicale; Aty,A. tamiyanavichii; P. micans,Prorocentrum micans) and local Alexandriumspecies Ach01. Sequences were obtained from the GenBank database usingthe keywords ‘Alexandrium LSU rDNA’ or ‘Alexandrium28S’. Phylogenetic trees were generated for an alignment of 81 sequences of641 bp in the D1/D2 region through Bayesian inference in MrBayes V3.2 and maximumlikelihood (ML) in PhyML. Bayesian analysis was carried out with 1 500 000 runs,with five separate initial trees. Convergence was checked through PSRF and AVSF.FigTree V1.3.1 was used as a visual representation of output trees. For MLanalysis these were carried out with 1000 bootstrap replicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526335&req=5

PLS033F3: Phylogenetic tree of 80 Alexandrium species andstrains (Atm, A. tamarense; Act, A. catenella;Afu, A. fundyense; Amn, A. minutum; Aaf,A. affine; Atr, A. tropicale; Aty,A. tamiyanavichii; P. micans,Prorocentrum micans) and local Alexandriumspecies Ach01. Sequences were obtained from the GenBank database usingthe keywords ‘Alexandrium LSU rDNA’ or ‘Alexandrium28S’. Phylogenetic trees were generated for an alignment of 81 sequences of641 bp in the D1/D2 region through Bayesian inference in MrBayes V3.2 and maximumlikelihood (ML) in PhyML. Bayesian analysis was carried out with 1 500 000 runs,with five separate initial trees. Convergence was checked through PSRF and AVSF.FigTree V1.3.1 was used as a visual representation of output trees. For MLanalysis these were carried out with 1000 bootstrap replicates.
Mentions: Using 81 sequences of length 641 bp, all aligning in the same D1/D2 LSU rDNA region,from different species of the genus Alexandrium and two strains ofP. micans, the phylogenetic distribution of localAlexandrium strains could be estimated (Fig. 3). Convergence of Bayesian trees was evaluatedthrough AVSF and PSRF. Values were less than 0.01 and ∼1 ±0.005,respectively, suggesting that the distribution reached a stationary phase in both datasets. Additionally, bootstrap values and the logarithm of the likelihood score of theoptimal tree (−4229.43342) extracted by maximum likelihood analysis wereindicative of a precise tree. The results indicate that the localAlexandrium strain is in Group I, dominated by A.tamarense, and is grouped with other previously sequencedAlexandrium strains from Chilean waters (ACC01, ACC02 and ACC07;Fig. 3). Fig. 3

Bottom Line: For species-specific determination, the intergenic spacer 1 (ITS1), 5.8S subunit, ITS2 and the hypervariable genomic regions D1-D5 of the large ribosomal subunit of local strains were sequenced and compared with two data sets of other Alexandrium sequences.Our results support the allocation of the Chilean Alexandrium species as a toxic Alexandrium tamarense rather than A. catenella, as currently defined.The results show that real-time PCR is useful to detect Alexandrium intake in filter-feeding molluscs.

View Article: PubMed Central - PubMed

Affiliation: FAVET-INBIOGEN, Faculty of Veterinary Sciences , University of Chile , Santa Rosa No 11.735 (PO 8820808), Santiago , Chile.

ABSTRACT

Background and aims: On the basis of morphological evidence, the species involved in South American Pacific coast harmful algal blooms (HABs) has been traditionally recognized as Alexandrium catenella (Dinophyceae). However, these observations have not been confirmed using evidence based on genomic sequence variability. Our principal objective was to accurately determine the species of Alexandrium involved in local HABs in order to implement a real-time polymerase chain reaction (PCR) assay for its rapid and easy detection on filter-feeding shellfish, such as mussels.

Methodology: For species-specific determination, the intergenic spacer 1 (ITS1), 5.8S subunit, ITS2 and the hypervariable genomic regions D1-D5 of the large ribosomal subunit of local strains were sequenced and compared with two data sets of other Alexandrium sequences. Species-specific primers were used to amplify signature sequences within the genomic DNA of the studied species by conventional and real-time PCR.

Principal results: Phylogenetic analysis determined that the Chilean strain falls into Group I of the tamarensis complex. Our results support the allocation of the Chilean Alexandrium species as a toxic Alexandrium tamarense rather than A. catenella, as currently defined. Once local species were determined to belong to Group I of the tamarensis complex, a highly sensitive and accurate real-time PCR procedure was developed to detect dinoflagellate presence in Mytilus spp. (Bivalvia) samples after being fed (challenged) in vitro with the Chilean Alexandrium strain. The results show that real-time PCR is useful to detect Alexandrium intake in filter-feeding molluscs.

Conclusions: It has been shown that the classification of local Alexandrium using morphological evidence is not very accurate. Molecular methods enabled the HAB dinoflagellate species of the Chilean coast to be assigned as A. tamarense rather than A. catenella. Real-time PCR analysis based on A. tamarense primers allowed the detection of dinoflagellate DNA in Mytilus spp. samples exposed to this alga. Through the specific assignment of dinoflagellate species involved in HABs, more reliable preventive policies can be implemented.

No MeSH data available.


Related in: MedlinePlus