MICROSCOPIA ELETRÔNICA DE VARREDURA COM TARDÍGRADOS (FILO TARDIGRADA)

SUGESTÃO DE PROTOCOLOS ADAPTADOS ÀS PECULIARIDADES DO GRUPO

Authors

  • Edivaldo L. Gomes Jr Universidade Federal de Pernambuco
  • Clélia M. C. da Rocha Universidade Federal Rural de Pernambuco

DOI:

https://doi.org/10.65259/rnzool.v9i2.52

Keywords:

Methodology, SEM, Taxonomy, Tardigrada

Abstract

Existing protocols for processing biological samples for scanning electron microscopy (SEM) usually do not produce good results when applied to tardigrades. This study aimed to test standard protocols for invertebrates in order to obtain better results with samples from this group. The two suggested protocols describe the preparation steps of the material, such as clamping, form of packaging of the specimens, rinses, post-fixation, dehydration, critical point drying and the manner of use of the SEM. In the "Protocol suggestion A" the material is fixed in 4% formaldehyde, rinses with sodium cacodilato buffer and distilled water, post-fixation with osmium tetroxide, dehydration with acetone, drying the sample to critical point with CO2, metallization in gold and the microscope configured in high vacuum mode. In the "Protocol suggestion B" the material is fixed in 70% ethanol, the samples are enclosed in capsules closed by meshes without washing steps and post-fixation. Dehydration is performed in ethanol and acetone, critical point drying with CO2, metallization in gold and the microscope configured to high vacuum mode.

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References

Amsellem, J. & Clément, P. 1980. A simplified method for the preparation of rotifers for transmission and scanning electron microscopy. Hydrobiologia 73: 119-122.

Baccetti, B. & F. Rosati .1969. Electron Microscopy on Tardigrades I. Connective tissue. Journal of submicroscopic cytology and pathology .,1: 197 - 205

Becquerel, P. 1950. La suspension de la vie au dessous de 1/20 K absolu par demagnetisation adiabatique de l'alun de fer dans le vide les plus elève. Comptes rendus de l'Académie des sciences ., Paris, 231: 261-263.

Bertolani, R.; R. Guidetti.; K.I. Jönsson .; T. Altiero.; D. Boschini & L. Rebecchi . 2004. Experiences on dormancy in tardigrades. Journal of Limnology, 63 Suppl.: 16-25.

Blaxter, M.; B. Elsworth & J. Daub. 2004. DNA taxonomy of a neglected animal phylum: an un expected diversity of tardigrades. Proceedings of the Royal of London Biological (Suppl.) 271: 189-192.

Castro, L.A.S.; R.M.V. Sanhueza.; R.F.F. Cantilillano & N.E.M. Rocha . 2002. Metodologia para observação da camada de cera em maçãs, utilizando microscopia eletrônica de varredura. Revista Brasileira de Fruticultura., Jaboticabal - SP, v. 24, n. 3, p. 774-775

Degma, P. 2010. Moss dwelling Tardigrada - from sampling to their identification; European Distributed Institute of Taxonomy EDIT Summer School. 35p.

Degma, P.; R. Bertolani & R. Guidetti. Actual checklist of Tardigrada species (2009-2015, Ver. 28: 31-03-2015). Disponível em world wide web em: http://www.tardigrada.modena.unimo.it/miscellanea/Actual%20checklist%20of%20Tardigrada.pdf .> Acesso em: Março de 2016.

Eibye-Jacobson, J. 2001. A new method for making SEM preparations of the tardigrade buccopharyngeal apparatus. Zoologischer Anzeiger- A Journal of Comparative Zoology, 240, 309–319. http://doi.org/10.1078/0044-5231-00038

Elmgren, R.1966. Baltic benthos communities and the role of meiofauna. Contr. Asko Lab. University of Stockholm. n.14, p.1-31.

Fontoura, P. & P. Morais . 2011. Assessment of traditional and geometric morphometrics for discriminating cryptic species of the Pseudechiniscus suillus complex (Tardigrada, Echiniscidae). Journal of Zoological Systematics and Evolutionary Research. doi: 10.1111/j.1439-0469.2010.00594.x

Garey, J.R; S.J. McInnes & P. Brent Nichols. 2008. Global diversity of tardigrades (Tardigrada) in freshwater. Hydrobiologia 595: 101 – 106.

Guil, N. & F.J. Cabrero-Sañudo. 2007. Analysis of the species description process for a little known invertebrate group: the limnoterrestrial tardigrades (Bilateria, Tardigrada). Biodiversity and Conservation.16: 1063 – 1086

Jorgensen, A.; T.M. Boesgaard .; N. Mobjerg. & R.M. Kristensen. 2014. The tardigrade fauna of Australian marine caves: With descriptions of nine new species of Arthrotardigrada. Zootaxa, 3802(4), 401–443. http://doi.org/10.11646/zootaxa.3802.4.1

Kinchin, I. M. 1994. The biology of tardigrades. London: Portland Press Ltd.186 p.

Hadju, E.; S. Peixinho & J.C.C. Fernandez. 2011. Identificação de esponjas 57-58. In: Esponjas marinhas da Bahia: Guia de campo e laboratório. Rio de Janeiro: Museu Nacional. 276p. ISBN: 978-85-7427-041-8

McInnes, S. J. 2001. Is It Real? Zoologischer Anzeiger., 240: 467 – 469

Melone, G. 1998. The rotifer corona by SEM. Hydrobiologia 387/388: 131-134

Michalczyk, L. & L. Kaczmarek. 2010. Description of Doryphoribius dawkinsi, a new species of tardigrada (Eutardigrada: Hypsibiidae) from the Costa Rican highlands, with the key to the genus Doryphoribius. Zootaxa, 58(2393), 46–58.

Mitchell, C. & W. Miller . 2008. A simple SEM (Scanning electron microscope) preparation protocol for tardígrados. Journal of the Pennsylvania Academy of science. 81 (2/3): 86-90

Nelson, D.R & N.J. Marley. 2000. The biology and ecology of lotic Tardigrada. Freshwater Biology 44: 93 - 108

Nelson, D. R. & R.P. Higgins. 1990. Tardigrada. In: DINDAL, D.L. Soil Biology Guide. New York: Ed. John Wiley and Sons. p 393-419.

Nelson, D.R. 2001. Ecology and classification of North American Freshwater invertebrates. 2nd ed. Academic press. p 527-550.

Nelson, D.R. & S.O. McInnes. 2002. Freshwater Meiofauna: Biology and Ecology, pp. 177-215.

Ono, F; M. Saigusa.; T. Uozomi.; Y. Matsushima.; H. Ikeda.; N.L. Saini & M. Yamashita . 2008. Effect of high hydrostatic pressure on to life of the tiny animal tardigrade. Journal of physics and chemistry of solids. 69; 2297-2300.

Pilato, G. 1981. Analisi di nuovi caratteri nello studio degli Eutardigradi. Animalia, 8: 51 – 57.

Pilato, G. & M.G. Binda .2010. Definition of families, subfamilies, genera and subgenera of the Eutardigrada, and keys to their identification. Zootaxa, 2404, 1-54.

Rahm, G. 1921. Biologische und physiologische Beitrãge zur Kenntinis der Moosfauna. Zeitschrift für Allgemeine Physiologie, 20: 1 - 35

Ramazzotti, G. & W. Maucci. 1983. Il phylum Tardigrada (III edição riveduta e aggiornata). Memorie de’ll Instituto Italiano di Idrobiologia. 41:1-1012.

Ramløv, H. & P. Westh . 2001. Cryptobiosis in the eutardigrade Adorybiotus (Richtersius) coronifer: tolerance to alcohols, temperature and de novo protein synthesis. Zoologischer Anzeiger. 240: 517 - 523

Rebecchi, L. & A. Guidi. 1991. First SEM studies on tardigrade spermatozoa. Invertebrate reproduction and Development, 19: 151 – 156.

Rebecchi, L; T. Altiero.; M. Cesari.; R. Bertolani.; A.M. Rizzo.; P.A. Corsetto & R. Guidetti. 2011. Resistance of the anhydrobiotic eutardigrade Paramacrobiotus richtersi to space flight (LIFE–TARSE mission on FOTON-M3). Journal of Zoological Systematics and Evolutionary Research. doi: 10.1111/j.1439-0469.2010.00606.x

Shively, S. & W. Miller. 2009. The use of HMDS (hexamethyldisilazane) to replace Critical Point Drying (CPD) in the preparation of tardigrades for SEM (Scanning Electron Microscope) imaging. Transactions of the Kansas academy of science Vol. 112, no. 3/4 p. 198-200

Silveira, M. 1989. Preparação de amostras biológicas para microscopia eletrônica de varredura. In: Manual sobre técnicas básicas em microscopia eletrônica. São Paulo: USP. v.1, p.71-79.

Tsujimoto, M.; S. Imura & H. Kanda. 2015. Recovery and reproduction of an Antarctic tardigrade retrieved from a moss sample frozen for over 30 years. Cryobiology. 1-4.

Wełnicz, W.; M. A. Grohme.; L. Kaczmarek .; R.O. Schill & M. Frohme. 2011. Anhydrobiosis in tardigrades-the last decade. Journal of Insect Physiology 57 (5),577–583.

Published

2015-06-25

How to Cite

Jr, E. L. G., & Rocha, C. M. C. da. (2015). MICROSCOPIA ELETRÔNICA DE VARREDURA COM TARDÍGRADOS (FILO TARDIGRADA): SUGESTÃO DE PROTOCOLOS ADAPTADOS ÀS PECULIARIDADES DO GRUPO. Revista Nordestina De Zoologia, 9(2), 16–32. https://doi.org/10.65259/rnzool.v9i2.52

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