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Chapter II 49 Frontiers in Zoology 2009, 6:3 http://www.frontiersinzoology.com/content/6/1/3 buffer (PB) for 2 hours or for 3–5 hours, and subsequently washed thrice with 0.1 M PB for 15 min each. The samples were stored in 0.1 M PB with 0.1% NaN 3 at 4°C. Material fixed for 2 hours was used for immunocytochemistry (ICC) and material fixed for 3–5 hours was used for scanning electron microscopy (SEM). Terebratalia transversa Adults were collected in the San Juan Archipelago, USA, in the vicinity of the Friday Harbor Laboratories, and were kept in running seawater tables. To obtain larvae, females were dissected and their eggs transferred into beaker glasses with filtered seawater. The seawater was changed several times in order to wash off follicle cells, and the eggs were left overnight for germinal vesicle breakdown. Males were opened and left in filtered seawater overnight. Thereafter, their testes were scraped out, macerated, and diluted with filtered seawater to obtain a sperm suspension. Prior to fertilization, sperm cells were activated by adding three drops of a 1 M Tris buffer solution (Sigma- Aldrich, St. Louis, MO, USA) to approximately 50 ml of sperm suspension. Larvae were maintained in embryo dishes at around 11°C and the filtered seawater was changed twice daily. Free swimming larvae, metamorphic stages, and juveniles five days after metamorphosis were relaxed in 7.14% MgCl 2 and fixed in 4% PFA in 0.1 M PB for 30 min at room temperature. Larvae were washed thrice for 15 min in 0.1 M PB and stored in 0.1 M PB with 0.1% NaN 3 at 4°C. Scanning electron microscopy For scanning electron microscopy (SEM), the specimens were postfixed in 1% OsO 4 , dehydrated in a graded acetone series, critical point dried, and sputter coated with gold. Digital images were acquired using a LEO 1430 VP SEM (Zeiss, Jena, Germany). Table 1: Comparative larval myoanatomy of the rhynchonelliform brachiopods Argyrotheca cordata, Terebratalia transversa, and A. cistellula Species Muscle Argyrotheca cordata Terebratalia transversa Argyrotheca cistellula Location Symbol in figures apical longitudinal muscles apical transversal muscle + + + apical lobe alm + + + (apical muscle ring) apical lobe atm central mantle muscles + + + (dorsal mantle muscles) mantle lobe empty arrowheads circular mantle muscle + + + (posterior muscle ring) mantle lobe arrows lateral mantle muscle - - + mantle lobe lmm longitudinal muscles + + - mantle and pedicle lobe lm pedicle muscles + + + pedicle lobe pm serial mantle muscles + + + mantle lobe double arrowsheads setae muscles + + - mantle lobe sm setae pouch musculature + + - mantle lobe arrowheads U-shaped muscle + + + (ventral mantle muscle) mantle lobe empty arrows Page 12 of 14 (page number not for citation purposes)
50 Chapter II Frontiers in Zoology 2009, 6:3 http://www.frontiersinzoology.com/content/6/1/3 F-actin labelling, confocal laserscanning microscopy (CLSM), and 3D reconstruction Prior to staining, larvae were washed thrice for 15 min in PB and incubated for 1 h in PB containing 0.1% Triton X- 100 (Sigma-Aldrich) to permeabilize the tissue. Then, the specimens were incubated in 1:40 diluted Alexa Fluor 488 phalloidin (Invitrogen, Molecular Probes, Eugene, OR, USA) and 3 μg/ml DAPI (Invitrogen) in the permeabilization solution overnight at 4°C. Subsequently, specimens were washed thrice for 15 min in 0.1 M PB and embedded in Fluoromount G (Southern Biotech, Birmingham, AL, USA) on glass slides. The same procedure was used for juveniles and adults, with the addition of a decalcifying step using 0.05 M EGTA (Sigma-Aldrich) at room temperature overnight prior to permeabilization and staining. Negative controls omitting the phalloidin dye were performed on all species in order to avoid potential misinterpretations caused by autofluorescence. The samples were analysed with a Leica DM RXE 6 TL fluorescence microscope equipped with a TCS SP2 AOBS laserscanning device (Leica Microsystems, Wetzlar, Germany). Animals were scanned at intervals of 0.49 μm or 0.64 μm, respectively, and the resulting image stacks were merged into maximum projection images. Photoshop CS3 (Adobe, San Jose, CA, USA) was used to create overlay images of CLSM and light micrographs and for assembling the figure plates. 3D reconstruction was performed on CLSM datasets using volume rendering algorithms of the graphics software Imaris 5.7.2 (Bitplane, Zurich, Switzerland). Competing interests The authors declare that they have no competing interests. Authors' contributions AA performed research and drafted the manuscript. AW designed and coordinated research, performed the SEM analysis, and contributed significantly to the writing of the manuscript. Both authors read and approved the final version of the manuscript. Additional material Additional file 1 Larval musculature of Argyrotheca cordata. Movie of a confocal scan through a fully developed larva of Argyrotheca cordata to illustrate the three-dimensional arrangement of the larval musculature. Click here for file [http://www.biomedcentral.com/content/supplementary/1742- 9994-6-3-S1.mpg] Acknowledgements We are grateful to Henrike Semmler (Copenhagen) for rearing and fixing Terebratalia larvae during the Comparative Invertebrate Embryology class 2006 at the Friday Harbor Laboratories and for comments on an early draft of the manuscript. We further thank the divers and the staff of the Marine Biological Station Banyuls-sur-mer for collecting the coralligène and for providing laboratory space. Scott Santagata (Brookville, New York) is thanked for comments on the manuscript and Jana Hoffmann (Berlin, Germany) for providing access to some of the classic literature. The valuable comments of an anonymous reviewer helped to improve the manuscript. This study was funded by the Danish Agency for Science, Technology and Innovation (grant no. 645-06-0294 to AW) and the Danish Research Agency (grant no. 21-04-0356 to AW). Research in the lab of A. Wanninger is further supported by the EU-funded Marie Curie Network MOLMORPH (contract grant number MEST-CT-2005-020542). References 1. Williams A, Rowell AJ: Evolution and Phylogeny. In Treatise on Invertebrate Paleontology, Part H, Brachiopoda Volume 1. Edited by: Moore RC. Lawrence, Kansas: The Geological Society of America and The University of Kansas; 1965:164-199. 2. Logan A: Geographic distribution of extant articulated brachiopods. In Treatise on Invertebrate Paleontology, Part H, Brachiopoda, Revised Volume 6. Edited by: Selden PA. Boulder, Colorado, and Lawrence, Kansas: The Geological Society of America, Inc. and The University of Kansas; 2007:3082-3115. 3. Stechmann A, Schlegel M: Analysis of the complete mitochondrial DNA sequence of the brachiopod Terebratulina retusa places Brachiopoda within the protostomes. Proc R Soc Lond B Biol Sci 1999, 266:2043-2052. 4. Williams A, Carlson SJ, Brunton CHC, Holmer LE, Popov L: A supraordinal classification of the Brachiopoda. Proc R Soc Lond B Biol Sci 1996, 351:1171-1193. 5. Nielsen C: The phylogenetic position of Entoprocta, Ectoprocta, Phoronida, and Brachiopoda. Integr Comp Biol 2002, 42:685-691. 6. Lüter C, Bartolomaeus T: The phylogenetic position of Brachiopoda – a comparison of morphological and molecular data. Zool Scr 1997, 26:245-253. 7. Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith SA, Seaver E, Rouse GW, Obst M, Edgecombe GD, et al.: Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 2008, 452:745-749. 8. Helmkampf M, Bruchhaus I, Hausdorf B: Multigene analysis of lophophorate and chaetognath phylogenetic relationships. Mol Phylogenet Evol 2008, 46:206-214. 9. Mallatt J, Winchell CJ: Testing the new animal phylogeny: First use of combined large-subunit and small-subunit rRNA gene sequences to classify the protostomes. Mol Biol Evol 2002, 19:289-301. 10. Passamaneck Y, Halanych KM: Lophotrochozoan phylogeny assessed with LSU and SSU data: Evidence of lophophorate polyphyly. Mol Phylogenet Evol 2006, 40:20-28. 11. Peterson KJ, Eernisse DJ: Animal phylogeny and the ancestry of bilaterians: inferences from morphology and 18S rDNA gene sequences. Evol Dev 2001, 3:170-205. 12. Cohen BL: Monophyly of brachiopods and phoronids: reconciliation of molecular evidence with Linnaean classification (the subphylum Phoroniformea nov.). Proc R Soc Lond B Biol Sci 2000, 267:225-231. 13. Cohen BL, Weydmann A: Molecular evidence that phoronids are a subtaxon of brachiopods (Brachiopoda: Phoronata) and that genetic divergence of metazoan phyla began long before the early Cambrian. Org Divers Evol 2005, 5:253-273. 14. Kowalevski AO: Observations sur le développement des brachiopodes (Analyse par Oehlert et Deniker). Arch Zool Exp Gen 1883, Series 2:57-76. Page 13 of 14 (page number not for citation purposes)
Page 1 and 2: Comparative neurogenesis, muscle de
Page 3 and 4: 2 Principal supervisor Assoc. P
Page 5 and 6: 4 Preface This thesis presents
Page 7 and 8: 6 Abstract Brachiopods are a sm
Page 9 and 10: 8 Acknowledgements The endeavou
Page 11 and 12: 10 Introduction Nervous system Micr
Page 13 and 14: 12 Material and methods Material an
Page 15 and 16: 14 Material and methods purpuratus,
Page 17 and 18: 16 Results and discussion Results a
Page 19 and 20: 18 Results and discussion posterior
Page 21 and 22: 20 Results and discussion Myogenesi
Page 23 and 24: 22 Results and discussion Wanninger
Page 25 and 26: 24 Results and discussion Growth pa
Page 27 and 28: 26 Results and discussion argument
Page 29 and 30: 28 References References Abdelkhale
Page 31 and 32: 30 References priapulids and protos
Page 33 and 34: 32 References James, M. A., Ansell,
Page 35 and 36: 34 References regionalization, prol
Page 37 and 38: 36 References 211. Williams, A., Ca
Page 39 and 40: 38 Chapter II Frontiers in Zoology
Page 49: 48 Chapter II Frontiers in Zoology
Page 53 and 54: 52 Chapter III Chapter III Altenbur
Page 55 and 56: 54 Chapter III Altenburger and Wann
Page 63 and 64: 62 Chapter IV Chapter IV Altenburge
Page 65 and 66: 64 Submitted manuscript Chapter IV

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