• Journal of the korean society of oceanography
• /
• v.34 no.3
• /
• pp.167-171
• /
• 1999

Since toxic Alexandrium catenella and non-toxic A. fraterculus are morphologically similar, they are difficult to discriminate under the light microscope. However, a novel technology, such as fluorescein isothiocyanate (FITC)-conjugated lectin probes enables easy and rapid differentiation. Toxic A. catenella bound seven different lectins, whereas the non-toxic A. fratercuzus did not bind Arachis hypogaea (PNA) lectin. In addition, Pseudo-nitrschia species in this study were also difficult to identify to species level with light microscope techniques, but it was possible to classify them using fluorescent lectins. Pseudo-nitzschia multistriata, P. subfraudulenta and P. pungens bound Canavalia ensiformis (ConA), whereas P. subpaclfica did not, and P. pungens also bound Ricinus communis (RCA). These results imply that lectin could be used as a critical tool in the differentiation of P. multistriata, P. subfraudulenta and P. pungens. However, P. subpacifica was not differentiated by the lectins tested. Therefore, it isconcluded that lectin probes are useful for discriminating toxic A. catenella from non-toxic A. fraterculus, and for the identification of some Pseudo-nitzschia species. In addition, this method has a great potential to speed and detection between non-toxic and toxic harmful algal blooms (HABs) in Korean biotoxin monitoring systems.

• Korean Journal of Environmental Biology
• /
• v.40 no.3
• /
• pp.290-300
• /
• 2022

A strain of Alexandrium species was established by isolating cells from Jangmok Bay, Korea. Its morphology and molecular phylogeny based on LSU rRNA gene sequences were examined. In addition, growth responses of this Alexandrium species to changes in temperature, salinity, and nutrient concentrations were investigated. This Alexandrium species from Jangmok Bay had a ventral pore on the 1', which was morphologically consistent with previously described Alexandrium tamarense and A. catenella. Phylogenetic analyses revealed that this isolate was assigned to A. pacificum (Group IV) within A. tamarense species complex. In growth experiments, relatively high growth rates and cell densities of A. pacificum (Group IV) were observed at 15℃ and 20℃. This species also grew under a wide range of salinity. This indicates that this Korean isolate of A. pacificum (Group IV) is a stenothermic and euryhaline species. In growth responses to changes in nutrient levels, enhanced growth rates and cell densities of A. pacificum(Group IV) were observed with additions of nitrate and phosphate. In particular, rapid uptakes of phosphate by A. pacificum (Group IV) were observed in experimental treatments, indicating that the increase in phosphate concentration could stimulate the growth of A. pacificum(Group IV).

• Proceedings of the Korean Aquaculture Society Conference
• /
• 2003.10a
• /
• pp.35-35
• /
• 2003

We, for the first time, reported molecular sequences of large subunit ribosomal DNA Dl-D3 region of A. hiranoi, A. leei and A. satoanum hitherto unreported. In addition, this study presented the full-length sequences of A. affine, A. fraterculus, A. catenella and A. tamarense occurring in Korean coastal waters. In total, 17 Alexandrium morphospecies were subjected to the phylogenetic analysis using the Maximum-likelihood (ML) method. The alignment result of sequences of A. hiranoi and A. pseudogonyaulax showed that there were only two substitutions without length heterogeneity implying their genetic affiliation. In ML tree, A. leei formed a deeply diverging branch probably because of the accelerated evolutionary rate, and its phylogenetic position was so ambiguous to resolve the phylogenetic relationship to the residual taxa. An A. satoanum culture showing morphological variation in the sulcal plate formed an independent divergent branch with consistent sister relationship to A. hiranoi/A. pseudogonyaulax clade supported by the high posterior probability (PP) value. Blast search in GenBank showed the sequence data of A. affine, A. fraterculus, A. catenella and A. tamarense corresponded to their morphological species designation. In ML tree, Alexandrium species were commonly split into four main clades. The inter-clade relationships were not clear and usually supported by the week PP values. In general, the sulcal plate of Alexandrium species seemed to reflect the true phylogeny at the main clade level, and the connection between the 1 and the apical pore complex seemed to reflect the phylogeny at the subclade level.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
• /
• v.3 no.2
• /
• pp.90-93
• /
• 1998

Cultured isolates of Prorocentrum minimum, P. micans, P. triestinum, P. balticum, Gymnodinium sanguineum, Alexandrium catenella, Scrippsiella trochoidea, and Heterosigma akashiwo from red tides in southern coast of Korea were phylogenetically compared on the basis of their 24S rRNA genes. For each isolate approximately 700 bp of 24S rDNA, encompassing D1 and D2 hypervariable domains, was amplified using the polymerase chain reaction and sequenced. The sequences were aligned with those reported in Genbank by using ClustalW program and phylogenetic tree was generated to show that morphospecies designations in the Alexandrium and Prorocentrum species are congruent with terminal taxa defined by phylogenetic analysis of the 24S rRNA fragment. Our results suggest that the molecular phylogeny approach could facilitate identification of domestic dinoflagellates which have ambiguous morphologies.

• Journal of Life Science
• /
• v.12 no.3
• /
• pp.250-255
• /
• 2002

Fluorescent species-specific DNA probe (AT1) of toxic dinoflagellate Arexandrium tamarense was tested on several other species, on comparison of binding activity at different preservatives for fixation of the cells, at different culture age and estimation of cell density by light microscope or epifluorescent microscope using whole cell hybridization. Th AT1 probe specifically bound to Alexandrium tamarense, whereas it did not bind to other phytoplankton, in particular Alexandrium catenella, morphologically similar to Alexandrium tamarense, could not react to AT1 probe. When cells were fixed with all three preservatives, labeling cells of Alexandrium tamarense emitted strong fluorescent signal intensity. In addition, regardless culture days, binding activity with AT1 probe was strong. The tell densities estimated by epifluorescent microscope were than those estimated by light microscope. The enumeration and identifying of Arexandriurn tamarense using DNA probe method will be contributed to a new biotoxin monitoring and prediction system in field.

• ALGAE
• /
• v.36 no.4
• /
• pp.299-314
• /
• 2021

Some species in the dinoflagellate genus Alexandrium are bioluminescent. Of the 33 formally described Alexandrium species, the bioluminescence capability of only nine species have been tested, and eight have been reported to be bioluminescent. The present study investigated the bioluminescence capability of seven Alexandrium species that had not been tested. Alexandrium mediterraneum, A. pohangense, and A. tamutum were bioluminescent, but A. andersonii, A. hiranoi, A. insuetum, and A. pseudogonyaulax were not. We also measured the bioluminescent intensity of A. affine, A. fraterculus, A. mediterraneum, A. ostenfeldii, A. pacificum, A. pohangense, A. tamarense, and A. tamutum. The mean 200-second-integrated bioluminescence intensity per cell ranged from 0.02 to 32.2 × 10⁴ relative luminescence unit per cell (RLU cell^-1), and the mean maximum bioluminescence intensity per cell per second (BL_Max) ranged from 0.01 to 10.3 × 10⁴ RLU cell^-1 s^-1. BL_Max was significantly correlated with the maximum growth rates of Alexandrium species, except for A. tamarense. A phylogenetic tree based on large subunit ribosomal DNA (LSU rDNA) showed that the bioluminescent species A. affine, A. catenella, A. fraterculus, A. mediterraneum, A. pacificum, and A. tamarense formed a large clade. However, the toxicity or mixotrophic capability of these species was split. Thus, their bioluminescence capability in this clade was more consistent than their toxicity or mixotrophic capability. Phylogenetic trees based on LSU rDNA and the luciferase gene of Alexandrium were consistent except for A. pohangense. The results of the present study can provide a basis for understanding the interspecific diversity in bioluminescence of Alexandrium.

• Proceedings of the Korean Aquaculture Society Conference
• /
• 2003.10a
• /
• pp.128-128
• /
• 2003

The annually outbreak of paralytic shellfish poisoning (PSP) were caused by toxic dinolagellate A. tamarense and A. catenella in Korea. The purpose of this study were to investigate the distribution of PSP-causative organisms, A. tamarense and A. catenella and their species classification. Sediment (Saemangeum, the south open sea) and water samples (southeastern coast) were sampled to establish clonal isolates in 2003. After isolation and purification, strains were cultured under $17^{\circ}C$, f/2 media, 14:10=L:D cycle. PST analysis and species identification were performed by HPLC-FD method and specific DNA probe, respectively. Thirty-ons strains were isolated from the Saemangeum reclamation, southeastern coast including Jinhae Bay and south open sea. PSTs were detected in all cultured strains. In eight strains from south offshore, major toxin components are GTX5, C1/2 and minors are GTX3/4, dcGTX3, neoSTX. Sixteen strains from south coastal area have GTX1/4, neoSTX, C1/2 as major toxin components and GTX2/3 as minors. Seven strains from the Saemangeum reclamation have GTX5, C1/2 as major toxin components and GTX1/2/3/4 as minors. Thus, among eight south offshore isolates, four A. tamarense have more toxic (38.31~l19.16 fmol.$cell^{-1}$) than A. catenella (3.78~13.13 fmol.$cell^{-1}$). With the previous results of different toxin composition, toxin components and toxin contents, .it is toxin profile that could used to diagnosis of regional toxic population and geographical distribution of both A. tamarense and A. catenella and their toxigenic strains.

• Fisheries and Aquatic Sciences
• /
• v.4 no.3
• /
• pp.120-129
• /
• 2001

Thirty-six dinoflagellate cysts, representing 15 genera were identified in the surface sediments obtained from the northwestern East China Sea. Three cyst morphotypes found in this survey have not previously been described in the East China Sea and adjacent waters: Seleno­pemphix sp. 2, Selenopemphix sp. 3 and Trinovantedinium sp. 1. In the northwestern East China Sea, Operculodinium centrocarpum, Spiniferites bulloideus and ellipsoidal cysts of Alexandrium were commonly observed. Moreover, it was recognized that the ellipsoidal cysts of Alexandrium, whose motile cells of A tamarense and/or A catenella are responsible to paralytic shellfish poisoning, distributed not only restricted to the coastal areas but also to the offshore stations far from the Changjiang River mouth.

• Proceedings of the Korean Aquaculture Society Conference
• /
• 2003.10a
• /
• pp.129-129
• /
• 2003

해양저질에서 휴면포자의 분포와 분포량에 대한 조사는 미래의 와편모조류 적조발생 시기나 장소를 예측할 수 있게 한다. 그러므로 저질내의 휴면포자를 찾고 분리하는 기술이 기본적으로 수행되어야 하며 지금까지 여러 방법이 개발되어 왔다. 이 연구의 목적은 기존의 휴면포자 계수방법들을 비교함으로써 연구의 효율을 제고 하고자 하는데 있다. 이 실험에서는 새만금 해역의 동일정점 저질내에 존재하는 휴면포자를 형광 염색하여 계수하는 Primuline 염색법, 해수, 휴면포자, sodium polytungstate의 비중차이를 이용하여 밀도구배원심법으로 분리하는 방법, 그리고 10% HCl로 탄산질을 제거하고 47% HF로 규산질을 제거한 뒤 휴면포자를 계수하는 고전적 계수방법 (palynological technique)을 사용하여 저질 내의 휴면포자를 분리 비교하였다. Primuline 염색법을 이용하였을 경우 Alexandrium famarense/catenella complex, Alexandrium sp. (spherical), Gonyaulax verior, Gonyaulax scrippsea, Gonyaulax spinifera complex등의 일부 종만 염색되어 관찰되었다. sodium polytungstate를 비중 1.3 g$cm^{-3}$으로 조정한 후 휴면포자를 분리한 밀도구배 원심법은 생물에 거의 무해한 것으로 알려져 있어서 발아가 가능해 휴면포자와 영양세포간의 관계를 규명할 수 있다는 장점이 있으나 계수에 있어서는 재현성이 떨어졌다. 반면 Palynological technique은 발아는 되지 않지만 석회질 벽을 가지는 휴면포자를 제외한 대부분의 종들이 관찰되었다. 이 결과는 실험 목적별로 휴면포자 계수방법을 달리 할 수 있음을 보여준다. 즉 발아실험을 할 경우에는 밀도구배원심법을, Alexandrium과 같은 특정종의 계수목적시에는 Primuline 염색법을, 정점 내 정확한 종 분포 파악을 위해서는 palynological technique을 사용하는 것이 효과적일 수 있다. 하지만 종분포 파악시에는 시간이 많이 소요되므로 좀더 신속하고 정확한 실험을 위해 분자생물학적인 기법들이 요구된다.

• Fisheries and Aquatic Sciences
• /
• v.7 no.4
• /
• pp.175-183
• /
• 2004

The dinoflagellate Alexandrium tamiyavanichii Balech, a producer of toxins causing paralytic shellfish poisoning (PSP), has recently been considered as one of main organisms responsible for toxication of shellfish in Japan. In this study, A. tamiyavanichii was subjected to a molecular phylogenetic analysis inferred from 28S rDNA D1-D2 sequences and a species-specific LSU rRNA-targeted oligonucleotide DNA probe was designed to identify A. tamiyavanichii using the whole cell-FISH (fluorescence in situ hybridization). The sequences of the 28S rDNA D1-D2 region of A. tamiyavanichii showed no difference from A. cohorticular AF1746l4 (present name A. tamiyavanichii) and formed a distinct clade from the 'tamarensis species complex'. The probe, TAMID2, reacted specifically with A. tamiyavanichii cultured cells, without any cross-reaction with other species belonging to the same genus, including A. tamarense, A. catenella, A. affine, A. fraterculus, A. insuetum and A. pseudogonyaulax. In a test of cross-reactivity with a field sample, TAMID2 reacted consistently with only A. tamiyavanichii, indicating that the present protocol involving the TAMID2 probe might be useful for detecting toxic A. tamiyavanichii in a simple and rapid manner.