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March 17, Srivastava, Sanjay, et al. January 9, Elizaga, Raquel Sosa January 9, February 11, Chakravarti, Uma, et al. To enhance your experience on our site, SAGE stores cookies on your computer. By continuing to use this site you consent to receive cookies. Remember me. Forgotten your password? Need help? Contact SAGE. Numeration was performed on a fraction of each water sample usually 1 mL, i.
Phytoplankton identification was carried out according to the following references: Cupp, ; Davis, ; Shirota, ; Yamaji, ; Tomas, ; Praseno and Sugestiningsih, ; Taylor et al. Total cyst extracts 10—16 mL obtained as described above were centrifuged at 12, rpm for 10 min at room temperature, and supernatants were discarded. The content of the glass bead tubes was examined under an inverted microscope to check if cyst walls had ruptured correctly.
To assess the presence of M. Amplification products were diluted with ultrapure H 2 O and underwent a second round of PCR using a pair of nested primers. Reactions were prepared as described above, using 0. Amplification success was verified by gel electrophoresis with 1. Amplicons were excised from the electrophoresis gel and purified using the QIAquick gel extraction kit Qiagen. Positive controls consisted of synthetic fragments Eurofins Genomics of the partial D1—D2 region from the following M. Table 1.
Primers used for M. To determine the LSU sequences from four to five colonies of the eight monoclonal cultures obtained in October , cells of strains LM5D, , , , , , LM2D and LM2D were placed in distilled water to disrupt cells. Phylogenetic positions of sequences obtained from Lampung Bay were inferred from neighbor-joining NJ and maximum likelihood ML analyses calculated in Mega 6 Tamura et al. Sequences obtained from motile cells and from cyst extracts were aligned with those of M.
Two C. Bootstrap support values for the NJ and ML trees were calculated in replicates. The resulting consensus tree with the highest likelihood was obtained from a heuristic search by applying NJ and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach. The rate variation model allowed for some sites to be evolutionarily invariable and the tree was drawn to scale, with branch lengths reflecting the number of substitutions per site. The analysis involved 46 nucleotide sequences 35 from GenBank database and 11 obtained from this study , for a total of positions.
NJ and ML analyses led to the same tree topology. In May , the presence of M. Cell concentrations varied from 2 to Both single cells, two-cell and four-cell chains were observed Figures 2a—c. However, the vast majority of motile cells were four-cell chains. In August , temporary cysts, or hyaline cysts, were also found in the water column during a M. Hyaline cysts were transparent, surrounded by a hyaline membrane, without chloroplasts and contained two to three red accumulation bodies and several spherical transparent bodies Figure 2d.
A paracingulum-like furrow structure was observed at the surface of hyaline cysts Figure 2e. These hyaline cysts were associated with four-cell chains. Figure 2.
Margalefidinium planktonic cell and cyst morphotypes observed at Lampung Bay. Margalefidinium polykrikoides cysts were detected in 17 of the 21 stations investigated in May , and three main morphotypes were observed Figures 2f—i. The first morphotype was quite similar to that described by Matsuoka and Matsuoka and Fukuyo , Its reticulated wall displayed a machichorate membranous ornamentation combined with thick evexate processes Figures 2f,g. This greenish-brown color morphotype presented a thin cyst wall with maybe two layers with a smooth surface.
In many cases, it exhibited a microgranular ornamentation. Archeopyle was not visible. This morphotype with a rough surface matched with the description of Tang and Gobler ; Figure 2h.
A variant previously described by Thoha et al. Its cyst wall was formed by a double layer with a microgranular membranous ornamentation and a mucilaginous coating around the cyst. The cyst contained a yellow-red accumulation body Figure 2i.
At least one M. The highest abundances of M. Table 2. Relative abundance of the three morphotypes observed for M. Regarding cyst extracts, only three of them could be reliably amplified and sequenced D1—D2 region, — bp , corresponding to stations D2 St1, D2 St2 and D3 St3. These amplicons were obtained with the primer combination D1R-Cpoly-R3. In order to compare them, the 11 sequences were aligned only on their common portion, representing a fragment of nearly bp. The phylogenetic analysis, conducted with 35 already published sequences representing the four ribotypes identified thus far East-Asian, Philippines, American-Malaysian, and Mediterranean clades , showed that the new sequences belonged to two different groups: those obtained from water samples clustered in the American-Malaysian clade, and sequences obtained from cyst extracts were closely related to the Mediterranean ribotype Figure 3.
The tree topology, supported by high bootstrap values, indicated a clear separation between the American-Malaysian, Philippines and East-Asian ribotypes, whereas the so-called Mediterranean ribotype tended to form a subgroup within the East-Asian clade.
Figure 3. Sequences obtained in this study are highlighted in black boxes. In the only survey performed in this area, no M. The present study is the first one that unequivocally reports the presence of the species M. Moreover, M. Many studies have pointed out that cysts play a decisive role in the ecology, population dynamics and spread of dinoflagellate species Genovesi-Giunti et al. However, the fact that significant densities of M. On the other hand, vegetative cells from Korean populations with an East-Asian ribotype have been shown to form temporary cysts surrounded by a hyaline membrane under certain environmental conditions Kim et al.
In the present study, hyaline cysts of M. This transient form, capable of rapidly turning into vegetative cells by means of a luminous stimulus, seems to be a strategy for maintaining populations during blooms Shin et al. Among the three main cyst morphotypes that were observed, the T-like one was predominant in sediments with the highest M. Such a mucilaginous layer around thornless morphotypes may allow the cysts to aggregate with other particles and thus become denser Bravo and Figueroa, As was shown for other taxonomic groups, cyst morphological changes can be considered as indicators of environmental variations e.
D2 St1, 2, 3, D2 St8, and D3 St6—7, which are the most affected by regular blooms, correspond to shallow and highly anthropized stations, very close to the shore and river mouths. At these stations, local hypoxic conditions in the bottom sediment due to terrestrial organic inputs and freshening are quite common Damar, ; Santoso and Hayami, Thus, the hypothesis that morphotype varies according to geochemical conditions in the water column during cyst formation and its residence time in the surface sediment is also plausible here.
Ornamented cysts could therefore be more recent or generated under conditions that do not inhibit the formation of the reticulated wall with membranous ornamentation. No ornamented cyst morphotype with thick spines and reticulate ornaments as described by Li et al. Obtaining vegetative cells from cyst germination is an essential step in the taxonomic identification of species with a dormant stage. In most studies describing M. As far as we know, only two studies have been able to obtain cyst germination from environmental samples and to formally identify the species: Mohamed and Al-Shehri and Li et al.
The most widely reported cyst morphotype is the MF one described by Matsuoka and Matsuoka and Fukuyo Wang et al. This ornamented morphotype was considered as the resting stage corresponding to M. However, different morphotypes were isolated from Korean sediment Kim et al.
Kim et al. Furthermore, Tang and Gobler generated spherical resting cysts from in vitro sexual reproduction of American-compatible strains and Hattenrath-Lehmann et al. Therefore, M. So far, only few studies could establish a clear relationship between morphotype and ribotype, and we cannot rule out that the different cyst morphotypes observed could also belong to several species which would then be regarded as cryptic.
This could be supported by the facts that Margalefidinium fulvescens has been formally described in Hurun bay Iwataki et al. The taxonomic clarification of this genus will require more investigations, as was the case within Alexandrium genus for Alexandrium catenella, Alexandrium tamarense, Alexandrium fundyense , and Alexandrium pacificum John et al.
Molecular detection of M. The American-Malaysian ribotype was detected from motile cells isolated in the water column in October The Mediterranean ribotype was found in three cyst extracts from sediment harvested from locations where several M. However, our results do not allow to assign a cyst morphotype to a ribotype because of the unsuccessful germination.
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On the other hand, in spite of our efforts, only three sequences from sediment cyst extracts could be obtained, all belonging to the Mediterranean ribotype. Considering the difficulty to amplify single sequences from sediment cyst extracts, mainly because of the existence of multiple PCR inhibitors Park et al. Though it is not devoid of experimental biases e.
We intend to apply this method on both sediment and water samples in the future studies on Lampung Bay. The phylogenetic analysis suggested that the so-called Mediterranean ribotype was rather a subgroup of the East-Asian cluster than a specific clade. Only sampling efforts and ribotyping of many individuals vegetative cells, hyaline, and resting cysts and of environmental samples will make it possible to understand the structuration of the ribotypes within this species. This approach was used to describe the diversity within the A. Our findings are comparable to those reported by Park et al.
Nagai et al. They highlighted three major populations and hypothesized contamination by pearl oyster transport during the —s. A Japanese pearl oyster Pinctada maxima farm was established in at station D3 St4 over an area of ha.
Thus, in addition to ballast water discharge, shellfish transfers can be a source of contamination, and further analysis is needed to test the hypothesis of an expansion through shellfish transfers in Lampung Bay. HT and AR revised the draft of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Mrs. Nurul Fitriya, Sugestiningsih, Trimaningsih, and Elly Asnaryanti for the phytoplankton analysis and their help in the field sampling, as well as Mrs. We also thank Mrs. Indriyani Anggi and Safira Qisthina for their help. We are grateful to Dr. Zainal and Dr. Dirhamsyah for their constant and full support. Aditya, V. Public awareness on harmful algal bloom HAB in Lampung bay. Al-Kandari, M. Kuwait: Kuwait Institute for Scientifc Research. Google Scholar. Al-Yamani, F. Kuwait: Kuwait Institute for Scientific Research. Azanza, R. An extensive Cochlodinium bloom along the western coast of palawan, Philippines.
Harmful Algae 7, — Bolch, C. Harmful Algae 6, — Bravo, I. Towards an ecological understanding of dinoflagellate cyst functions. Microorganisms 2, 11— Corrales, R. Impacts of harmful algae on seafarming in the Asia-Pacific areas. Cupp, E. Damar, A. The current president, Joko Widodo , is the seventh president since independence, and the first who does not have a high-ranking political or military background. Indonesia is one of the world's largest democracies and the most populous Muslim-majority country.
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