Cryptomonads are unicellular, biflagellate algae. Generally, cryptomonad cells cannot be preserved well because of their fragile nature, and an improved methodology should be developed to identify cryptomonads from natural habitats. In this study, we tried using several cytological fixatives, includ...
Cryptomonads are unicellular, biflagellate algae. Generally, cryptomonad cells cannot be preserved well because of their fragile nature, and an improved methodology should be developed to identify cryptomonads from natural habitats. In this study, we tried using several cytological fixatives, including glutaraldehyde, formaldehyde, and their combinations to preserve field samples collected from various waters, and the currently used fixative, Lugol's solution was tested for comparison. Results showed that among the fixatives tested, glutaraldehyde preserved the samples best, and the optimal concentration of glutaraldehyde was 2%. The cell morphology was well preserved by glutaraldehyde. Cells kept their original color, volume, and shape, and important taxonomic features such as furrow/gullet complex, ejectosomes, as well as flagella could be observed clearly, whereas these organelles frequently disappeared in Lugol's solution preserved samples. The osmotic adjustments and buffers tested could not preserve cell density significantly higher. Statistical calculation showed the cell density in the samples preserved by 2% glutaraldehyde remained stable after 43 days of the fixation procedure. In addition, DNA was extracted from glutaraldehyde preserved samples by grinding with liquid nitrogen and the 18S rDNA sequence was amplified by PCR. The sequence was virtually identical to the reference sequence, and phylogenetic analyses showed very close relationship between it and sequences from the same organism. To sum up, the present study demonstrated that 2% unbuffered glutaraldehyde, without osmotic adjustments, can preserve cryptomonads cells for identification, in terms of both light microscopy and phylogenetic analyses based on DNA sequences.
Cryptomonads are unicellular, biflagellate algae. Generally, cryptomonad cells cannot be preserved well because of their fragile nature, and an improved methodology should be developed to identify cryptomonads from natural habitats. In this study, we tried using several cytological fixatives, including glutaraldehyde, formaldehyde, and their combinations to preserve field samples collected from various waters, and the currently used fixative, Lugol's solution was tested for comparison. Results showed that among the fixatives tested, glutaraldehyde preserved the samples best, and the optimal concentration of glutaraldehyde was 2%. The cell morphology was well preserved by glutaraldehyde. Cells kept their original color, volume, and shape, and important taxonomic features such as furrow/gullet complex, ejectosomes, as well as flagella could be observed clearly, whereas these organelles frequently disappeared in Lugol's solution preserved samples. The osmotic adjustments and buffers tested could not preserve cell density significantly higher. Statistical calculation showed the cell density in the samples preserved by 2% glutaraldehyde remained stable after 43 days of the fixation procedure. In addition, DNA was extracted from glutaraldehyde preserved samples by grinding with liquid nitrogen and the 18S rDNA sequence was amplified by PCR. The sequence was virtually identical to the reference sequence, and phylogenetic analyses showed very close relationship between it and sequences from the same organism. To sum up, the present study demonstrated that 2% unbuffered glutaraldehyde, without osmotic adjustments, can preserve cryptomonads cells for identification, in terms of both light microscopy and phylogenetic analyses based on DNA sequences.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
대상 데이터
Cryptomonas pyrenoidifera was collected from a eutrophic fishpond (30°31'47''N, 114°21'16''E), where it bloomed.
Samples of Campylomonas reflexa were collected from Lake Nanhu (30°29'46''N, 114°20'12''E) in the city of Wuhan.
Samples of seven cryptomonad species were used in the present study. Samples were collected by phytoplankton nets from various kinds of water bodies in Hubei Province of China from January 2009 to July 2012. Samples of Campylomonas reflexa were collected from Lake Nanhu (30°29'46''N, 114°20'12''E) in the city of Wuhan.
The fixatives were prepared just before use. The samples of Campylomonas reflexa were used. After 24 h of fixation, cells were observed and counted under light microscopy.
The effects of buffers and osmotic adjustments were evaluated (Experiment C). Two buffers were tested: PBS (final concentration of 0.1 M, pH 7.2) and HEPES (Calbiochem; final concentration of 30 mM). Two osmotic adjustments were tested: sorbitol and sucrose (Sinopharm Chemical Reageal Company; saturated).
was collected from the reservoir of the Three Gorges Dam (31°07'21''N, 110°47'01''E), where it bloomed and the water presented a red-brown color.
were collected from Lake Donghu (30°32'55''N, 114°21'16''E).
데이터처리
To evaluate cell lysis caused by fixation, the cells were observed and counted for 53 days. ANOVA was carried out to test the difference in cell densities among the days.
One-way analysis of variance (ANOVA) was carried out to test the difference in cell densities among the treatments with a discrimination level of p < 0.05.
이론/모형
83) [43]. The alignments were refined manually by MEGA (ver. 4.0) [42], and phylogenetic trees were constructed using the neighbor-joining (NJ) method in the same software. Bootstrap analyses with 1,000 replicates were calculated to estimate statistical reliability.
성능/효과
In the samples fixed with 2% glutaraldehyde, there was no significant difference in cell density (p > 0.05) until 43 days after the fixation procedure.
More than 10 kinds of fixatives with different concentrations were tested to preserve cryptomonads samples in this study, such as paraformaldehyde, chloroform, acetone, ethanol, glacial acetic acid, methanol, ether, picronitric acid, and different combinations of them (data not shown), but none gave better results than glutaraldehyde with final concentration of 2%.
Phylogenetic analyses were performed. Results of the present study clearly showed that glutaraldehyde without any buffer or osmotic adjustment could preserve the morphology of cryptomonads well for light microscopy at the final concentration of 2%, and cell lysis due to fixation was negligible after 43 days of the fixation procedure. For the first time, DNA extraction and PCR amplification were successfully performed on cryptomonads samples preserved with glutaraldehyde.
The 18S rDNA sequences of Cryptomonas pyrenoidifera amplified from samples preserved with glutaraldehyde and from live cells were 1,548 bp long, and only one site was different between them. A T-C mutation happened.
The results of Experiment B showed that, after 24 h of fixation, the cell density in the Cryptomonas tetrapyrenoidifera samples fixed by 2% glutaraldehyde was significantly higher than that in samples fixed by glutaraldehyde with other concentrations (p < 0.05), and there was no significant difference between the cell density in samples fixed with 2% glutaraldehyde and in samples fixed with Lugol’s solution (p > 0.05).
There was no significant difference between the numbers of undistorted cell bodies in group F1 and groups G1, F2, and G2 (p > 0.05), but in groups G1 and G2, the number of cells retaining flagella was significantly higher than that in groups F1 and F2 (p < 0.05).
Meanwhile, fixation of phytoplankton has been performed mainly with formaldehyde for qualitative study [44], which is destructive to cryptomonads cells, as shown in this study and in previous works [26]. This study clearly demonstrated that fixation with 2% glutaraldehyde was practical for microscopy. First, the cell shapes were not altered, and neither swelling nor shrinking of cell volume happened; second, the cell kept its original color; and finally important taxonomic features such as ejectosomes, furrow/gullet, and flagella were retained.
참고문헌 (48)
Andreoli, C., C. Tolomio, N. Rascio, and R. Talarico. 1986. Some observations on a Cryptophyceae responsible for a winter red bloom. G. Bot. Ital. 120: 70-71.
Andersen, R. A. 1992. Diversity of eukaryotic algae. Biodivers. Conserv. 1: 267-292.
Auinger, B. M., K. Pfandl, and J. Boenigk. 2008. Improved methodology for identification of protists and microalgae from plankton samples preserved in Lugol's iodine solution: Combining microscopic analysis with single-cell PCR. Appl. Environ. Microbiol. 74: 2505-2510.
Cavalier-Smith, T., J. A. Couch, K. E. Thorsteinsen, P. Gilson, J. A. Deane, D. R. A. Hill, and G. I. McFadden. 1996. Cryptomonad nuclear and nucleomorph 18S rRNA phylogeny. Eur. J. Phycol. 31: 315-328.
Dame, R., M. Alber, D. Allen, M. Mallin, C. Montague, A. Lewitus, et al. 2000. Estuaries of the South Atlantic coast of North America: Their geographical signatures. Estuar. Coast. 23: 793-819.
Deane, J. A., I. M. Strachan, G. M. Saunders, D. R. A. Hill, and G. I. McFadden. 2002. Cryptomonad evolution: Nuclear 18S rDNA phylogeny versus cell morphology and pigmentation. J. Phycol. 38: 1236-1244.
Doughty, M. J., J. P. Bergmanson, and Y. Blocker. 1995. Impact of glutaraldehyde versus glutaraldehyde-formaldehyde fixative on cell organization in fish corneal epithelium. Tissue Cell 27: 701-712.
Gillot, M. 1990. Phylum Cryptophyta (Cryptomonads), pp. 139-151. In L. Margulis, J. O. Corliss, M. Melkonian, and D. J. Chapman (eds.). Handbook of Protoctista. Jones Bartlett Publishers, Boston.
Godhe, A., D. M. Anderson, and A. Rehnstam-Holm. 2002. PCR amplification of microalgal DNA for sequencing and species identification: Studies on fixatives and algal growth stages. Harmful Algae 1: 375-382.
Hoef-Emden, K. 2007. Revision of the genus Cryptomonas (Cryptophyceae) II: Incongruences between the classical morphospecies concept and molecular phylogeny in smaller pyrenoid-less cells. Phycologia 46: 402-428.
Hoef-Emden, K., B. Marin, and M. Melkonian. 2002. Nuclear and nucleomorph SSU rDNA phylogeny in the Cryptophyta and the evolution of cryptophyte diversity. J. Mol. Evol. 55: 161-179.
Hoef-Emden, K. and M. Melkonian. 2003. Revision of the genus Cryptomonas (Cryptophyceae) I: A combination of molecular phylogeny and morphology provides insights into a long hidden dimorphism. Protist 154: 371-409.
Hotzel, G. and R. Croome. 1999. A Phytoplankton Methods Manual for Australian Freshwaters. Land and Water Resources Research and Development Corporation, Green Words & Images, Canberra.
Jordan, W. P., M. V. Dahl, and H. L. Albert. 1972. Contact dermatitis from glutaraldehyde. Arch. Dermatol. 105: 94-95.
Katano, T., M. Yoshida, J. Lee, M. S. Han, and Y. Hayami. 2009. Fixation of Chattonella autiqua and C. marina (Raphidophyceae) using Hepes-buffered paraformaldehyde and glutaraldehyde for flow cytometry and light microscopy. Phycologia 48: 473-479.
Klaveness, D. 1988. Ecology of the Cryptomonadida: A first review. pp. 105-133. In C. D. Sandgren (ed.). Growth and Reproductive Strategies of Freshwater Phytoplankton. Cambridge University Press, New York.
Klaveness, D. 1988. Biology and ecology of the Cryptophyceae: Status and challenges. Biol. Oceanogr. 6: 257-270.
Kugrens, P. and B. L. Clay. 2003. Cryptomonads, pp. 715-755. In J. D. Wehr and R. G. Sheath (eds.). Freshwater Algae of North America. Academic Press, San Diego.
Leakey, R. J. G., P. K. Burkhill, and M. A. Sleigh. 1994. A comparion of fixatives for the estimation of abundance and biovolume of marine planktonic ciliate populations. J. Plankton Res. 16: 375-389.
Marin, I., A. Aguilera, B. Reguera, and J. P. Abad. 2001. Preparation of DNA suitable for PCR amplification from fresh or fixed single dinoflagellate cells. Biotechniques 30: 88-90.
Menden-Deuer, S., E. J. Lessard, and J. Satterberg. 2001. Effect of preservation on dinoflagellate and diatom cell volume and consequences for carbon biomall predictions. Mar. Ecol. Prog. Ser. 222: 41-50.
Menezes, M. and G. Novarino. 2003. How diverse are planktonic cryptomonads in Brazil? Advantages and difficulties of a taxonomic-biogeographical approach. Hydrobiologia 502: 297-306.
Monsan, P., G. Puzo, and H. Marzarguil. 1975. Etude du mecanisme d'etablissement des liaisons glutaraldehyde-proteines. Biochimie 57: 1281-1292.
Nishijima, T. 1990. Growth characteristics of Plagioselmis sp. (strain 87) causing freshwater red tide in the lower part of the Nakasuji River, Japan. Nippon Suisan Gakkaishi. 56: 353-359.
Paljarvi, L., J. H. Garcia, and H. Kalimo. 1979. The efficiency of aldehyde fixation for electron microscopy: Stabilization of rat brain tissue to withstand osmotic stress. Histochem. J. 11: 267-276.
Richlen, M. and P. Barber 2005. A technique for the rapid extraction of microalgal DNA from single live and preserved cells. Mol. Ecol. Notes 5: 688-691.
Santore, U. J. 1984. Some aspects of taxonomy in the Cryptophyceae. New Phycol. 98: 627-646.
Shiozawa, D., J. Kudo, R. P. Evans, S. R. Woodward, and R. N. Williams. 1992. DNA extraction from preserved trout tissues. West. N. Am. Naturalist 52: 29-34.
Stoecker, D. K., D. J. Gifford, and M. Putt. 1994. Preservation of marine planktonic ciliates: Losses and cell shrinkage during fixation. Mar. Ecol. Prog. Ser. 110: 293-299.
Tamura, K., J. Dudley, M. Nei, and S. Kumar. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599.
Thompson, J. D., T. J. Gibson, F. Plewniak, and D. G. Higgins. 1997. The CLUSTAL_X Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882.
Throndsen J. 1978. Preservation and storage, pp. 69-74. In A. Sournia (ed.). Phytoplankton Manual. United Nations Educational, Scientific and Cultural Organization, Paris.
Voo, K. S. and M. Britta. 1998. Extraction and amplifi-cation of mitochondrial DNA from formalin-fixed deep-sea mollusks. Biotechniques 24: 243-247.
Williams, C., F. Ponten, C. Moberg, P. Soderkvist, M. Uhlen, J. Ponten, et al. 1999. A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am. J. Pathol. 155: 1467-1471.
Woelfl, S. and B. A. Whitton. 2000. Sampling, preservation and quantification of biological samples from highly acidic environments ( $pH{\leq}3$ ). Hydrobiologia 433: 173-180.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.