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X-Ray Microscopy of the Larval Crustacean Brain

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Brain Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2047))

Abstract

Micro-computed X-ray tomography (μCT) coupled with visualization techniques such as three-dimensional reconstruction of internal morphological structures has opened up new pathways for analyzing the anatomy of nervous systems in intact specimens. The possibility for combining μCT with other techniques is one of the major advantages of μCT scanning, and the technical development of higher resolutions in lab-based μCT-scanners allows for investigating the anatomy of specimens in the sub-milimeter range. The European shore crab Carcinus maenas features a larval development over four zoeal and one megalopal stage with body lengths ranging from 500 μm to 2000 μm. The developing nervous system in the larvae of C. maenas is organized into a central brain which is connected via esophageal connectives with a ventral nerve chord and segmental ganglia. Since soft tissues such as the nervous tissues feature low contrasts compared to other tissues such as muscles or cuticularized body parts, the interpretation in μCT scans is challenging and needs some practice. The protocol described here is also applicable for larger specimens of a variety of species and spans over 2–3 days resulting in an image stack ready for postprocessing and visualization.

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References

  1. Holst S, Michalik P, Noske M, Krieger J, Sötje I (2016) Potential of X-ray micro-computed tomography for soft-bodied and gelatinous cnidarians with special emphasis on scyphozoan and cubozoan statoliths. J Plankton Res 38:1225–1242. https://doi.org/10.1093/plankt/fbw054

    Article  CAS  Google Scholar 

  2. Henne S, Friedrich F, Hammel JU, Sombke A, Schmidt-Rhaesa A (2016) Reconstructing the anterior part of the nervous system of Gordius aquaticus (Nematomorpha, cycloneuralia) by a multimethodological approach. J Morphol 278:106–118. https://doi.org/10.1002/jmor.20623

    Article  PubMed  Google Scholar 

  3. O’Sullivan JDB, Behnsen J, Starborg T, MacDonald AS, Phythian-Adams AT, Else KJ, Cruickshank SM, Withers PJ (2018) X-ray micro-computed tomography (μCT): an emerging opportunity in parasite imaging. Parasitology 145:1–7. https://doi.org/10.1017/s0031182017002074

    Article  Google Scholar 

  4. Dinley J, Hawkins L, Paterson G, Ball AD, Sinclair I, Sinnett-Jones P, Lanham S (2010) Micro-computed X-ray tomography: a new non-destructive method of assessing sectional, fly-through and 3D imaging of a soft-bodied marine worm. J Microsc 238:123–133. https://doi.org/10.1111/j.1365-2818.2009.03335.x

    Article  CAS  PubMed  Google Scholar 

  5. Faulwetter S, Vasileiadou A, Kouratoras M, Dailianis T, Arvanitidis C (2013) Micro-computed tomography: introducing new dimensions to taxonomy. ZooKeys 263:1–45. https://doi.org/10.3897/zookeys.263.4261

    Article  Google Scholar 

  6. Handschuh S, Baeumler N, Schwaha T, Ruthensteiner B (2013) A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario. Front Zool 10:44. https://doi.org/10.1186/1742-9994-10-44

    Article  PubMed  PubMed Central  Google Scholar 

  7. Akkari N, Enghoff H, Metscher BD (2015) A new dimension in documenting new species: high-detail imaging for myriapod taxonomy and first 3D cybertype of a new millipede species (Diplopoda, Julida, Julidae). PLoS One 10:e0135243. https://doi.org/10.1371/journal.pone.0135243

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Michalik P, Piacentini L, Lipke E, Ramirez M (2013) The enigmatic Otway odd-clawed spider (Progradungula otwayensis Milledge, 1997, Gradungulidae, Araneae): natural history, first description of the female and micro-computed tomography of the male palpal organ. ZooKeys 335:101–112. https://doi.org/10.3897/zookeys.335.6030

    Article  Google Scholar 

  9. Nischik ES, Krieger J (2018) Evaluation of standard imaging techniques and volumetric preservation of nervous tissue in genetically identical offspring of the crayfish Procambarus fallax cf. virginalis (Marmorkrebs). PeerJ 6:e5181. https://doi.org/10.7717/peerj.5181

    Article  PubMed  PubMed Central  Google Scholar 

  10. Sombke A, Lipke E, Michalik P, Uhl G, Harzsch S (2015) Potential and limitations of X-Ray micro-computed tomography in arthropod neuroanatomy: a methodological and comparative survey. J Comp Neurol 523:1281–1295. https://doi.org/10.1002/cne.23741

    Article  PubMed  PubMed Central  Google Scholar 

  11. Steinhoff POM, Sombke A, Liedtke J, Schneider JM, Harzsch S, Uhl G (2017) The synganglion of the jumping spider Marpissa muscosa (Arachnida: Salticidae): insights from histology, immunohistochemistry and microCT analysis. Arthropod Struct Dev 46:156–170. https://doi.org/10.1016/j.asd.2016.11.003

    Article  PubMed  Google Scholar 

  12. Metscher BD (2009) MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physiol 9:11. https://doi.org/10.1186/1472-6793-9-11

    Article  PubMed  PubMed Central  Google Scholar 

  13. Köhnk S, Baudewig J, Brandis D, Boretius S (2017) What’s in this crab? MRI providing high-resolution three-dimensional insights into recent finds and historical collections of Brachyura. Zoology 121:1–9. https://doi.org/10.1016/j.zool.2016.11.004

    Article  PubMed  Google Scholar 

  14. Hounsfield GN (1973) Computerized transverse axial scanning (tomography): part 1. Description of system. Br J Radiol 46:1016–1022. https://doi.org/10.1259/0007-1285-46-552-1016

    Article  CAS  PubMed  Google Scholar 

  15. Bucher D, Scholz M, Stetter M, Obermayer K, Pflüger H-J (2000) Correction methods for three-dimensional reconstructions from confocal images: I. Tissue shrinking and axial scaling. J Neurosci Methods 100:135–143. https://doi.org/10.1016/S0165-0270(00)00245-4

    Article  CAS  PubMed  Google Scholar 

  16. Huys R, Olesen JT, Petrunina A, Martin JW (2014) Tantulocarida. In: Martin JW, Olesen JT, Høeg JT (eds) Atlas of crustacean larvae. Johns Hopkins University Press, Baltimore, pp 122–127

    Google Scholar 

  17. Ahyong ST, Haug JT, Haug C (2014) Stomatopoda. In: Martin JW, Olesen JT, Høeg JT (eds) Atlas of crustacean larvae. Johns Hopkins University Press, Baltimore, pp 185–187

    Google Scholar 

  18. Anger K (2001) The biology of decapod crustacean larvae. AA Balkema Publishers, Rotterdam

    Google Scholar 

  19. Anger K (2006) Contributions of larval biology to crustacean research: a review. Invertebr Reprod Dev 49:175–205. https://doi.org/10.1080/07924259.2006.9652207

    Article  Google Scholar 

  20. Anger K, Queiroga H, Calado R (2015) Larval development and behaviour strategies in Brachyura. In: Castro P, Davie P, Guinot D, Schram F, von Vaupel Klein JC (eds) Treatise on zoology—anatomy, taxonomy, biology. The crustacea, volume 9, part C-I, Decapoda: Brachyura. Brill, Leiden, Boston, pp 317–374

    Google Scholar 

  21. Haug JT, Haug C (2015) “Crustacea”: comparative aspects of larval development. In: Wanninger A (ed) Evolutionary developmental biology of invertebrates 4: ecdysozoa II: crustacea. Springer Vienna, Vienna, pp 1–37

    Google Scholar 

  22. Martin JW (2014) Brachyura. In: Martin JW, Olesen JT, Høeg JT (eds) Atlas of crustacean larvae. Johns Hopkins University Press, Baltimore, pp 295–310

    Google Scholar 

  23. Carlton JT, Cohen AN (2003) Episodic global dispersal in shallow water marine organisms: the case history of the European shore crabs Carcinus maenas and C. aestuarii. J Biogeogr 30:1809–1820. https://doi.org/10.1111/j.1365-2699.2003.00962.x

    Article  Google Scholar 

  24. Cohen AN, Carlton JT, Fountain MC (1995) Introduction, dispersal and potential impacts of the green crab Carcinus maenas in San Francisco Bay, California. Mar Biol 122:225–237. https://doi.org/10.1007/BF00348935

    Article  Google Scholar 

  25. Grosholz ED, Ruiz GM (1995) Spread and potential impact of the recently introduced European green crab, Carcinus maenas, in central California. Mar Biol 122:239–247. https://doi.org/10.1007/BF00348936

    Article  Google Scholar 

  26. Hidalgo FJ, Barón PJ, Orensanz JM (2005) A prediction come true: the green crab invades the Patagonian coast. Biol Invasions 7:547–552. https://doi.org/10.1007/s10530-004-5452-3

    Article  Google Scholar 

  27. Yamada SB, Dumbauld BR, Kalin A, Hunt CE, Figlar-Barnes R, Randall A (2005) Growth and persistence of a recent invader Carcinus maenas in estuaries of the northeastern pacific. Biol Invasions 7:309–321. https://doi.org/10.1007/s10530-004-0877-2

    Article  Google Scholar 

  28. Young A, Elliott J (2018) Population dynamics of green crabs (Carcinus maenas)—a review. Preprints. https://doi.org/10.20944/preprints201807.0436.v2

  29. Epifanio CE, Cohen JH (2016) Behavioral adaptations in larvae of brachyuran crabs: a review. J Exp Mar Biol Ecol 482:85–105. https://doi.org/10.1016/j.jembe.2016.05.006

    Article  Google Scholar 

  30. Forward RB (2009) Larval biology of the crab Rhithropanopeus harrisii (Gould): a synthesis. Biol Bull 216:243–256. https://doi.org/10.1086/BBLv216n3p243

    Article  PubMed  Google Scholar 

  31. Spitzner F, Meth R, Krüger C, Nischik E, Eiler S, Sombke A, Torres G, Harzsch S (2018) An atlas of larval organogenesis in the European shore crab Carcinus maenas L. (Decapoda, Brachyura, Portunidae). Front Zool 15:27. https://doi.org/10.1186/s12983-018-0271-z

    Article  PubMed  PubMed Central  Google Scholar 

  32. Krieger J, Sombke A, Seefluth F, Kenning M, Hansson BS, Harzsch S (2012) Comparative brain architecture of the European shore crab Carcinus maenas (Brachyura) and the common hermit crab Pagurus bernhardus (Anomura) with notes on other marine hermit crabs. Cell Tissue Res 348:47–69. https://doi.org/10.1007/s00441-012-1353-4

    Article  PubMed  Google Scholar 

  33. Harzsch S, Dawirs RR (1993) On the morphology of the central nervous system in larval stages of Carcinus maenas L. (Decapoda, Brachyura). Helgolander Meeresunters 47:61–79. https://doi.org/10.1007/BF02366185

    Article  Google Scholar 

  34. Panel on Animal Health and Welfare (2005) Opinion of the scientific panel on Animal Health and Welfare (AHAW) on a request from the Commission related to the aspects of the biology and welfare of animals used for experimental and other scientific purposes. EFSA J 3:292. https://doi.org/10.2903/j.efsa.2005.292

    Article  Google Scholar 

  35. Jahn H, Oliveira IDS, Gross V, Martin C, Hipp A, Mayer G, Hammel JU (2018) Evaluation of contrasting techniques for X-ray imaging of velvet worms (Onychophora). J Microsc 270:343–358. https://doi.org/10.1111/jmi.12688

    Article  CAS  PubMed  Google Scholar 

  36. Gutiérrez Y, Ott D, Töpperwien M, Salditt T, Scherber C (2018) X-ray computed tomography and its potential in ecological research: a review of studies and optimization of specimen preparation. Ecol Evol 8:1–18. https://doi.org/10.1002/ece3.4149

    Article  Google Scholar 

  37. Betz O, Wegst U, Weide D, Heethoff M, Helfen L, Lee W-K, Cloetens P (2007) Imaging applications of synchrotron X-ray phase-contrast microtomography in biological morphology and biomaterials science. I. General aspects of the technique and its advantages in the analysis of millimetre-sized arthropod structure. J Microsc 227:51–71. https://doi.org/10.1111/j.1365-2818.2007.01785.x

    Article  PubMed  Google Scholar 

  38. Wipfler B, Pohl H, Yavorskaya MI, Beutel RG (2016) A review of methods for analysing insect structures—the role of morphology in the age of phylogenomics. Curr Opin Insect Sci 18:60–68. https://doi.org/10.1016/j.cois.2016.09.004

    Article  PubMed  Google Scholar 

  39. Lösel P, Heuveline V (2016) Enhancing a diffusion algorithm for 4D image segmentation using local information. In: Medical Imaging 2016: image processing. International Society for Optics and Photonics, Bellingham WA, p 97842L

    Google Scholar 

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Acknowledgments

We cordially thank Marie K. Hörnig for macrophotographs of specimen preparation as well as Steffen Harzsch for reviewing and constructive criticism of the first version of the manuscript. This work was supported by the German Science Foundation (Research Training Group 2010 RESPONSE, DFG INST 292/119-1 FUGG, and DFG INST 292/120-1 FUGG).

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Authors and Affiliations

  1. Department of Cytology and Evolutionary Biology, Zoological Institute and Museum, University of Greifswald, Greifswald, Germany

    Jakob Krieger & Franziska Spitzner

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  1. Jakob Krieger
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  2. Franziska Spitzner
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Correspondence to Jakob Krieger .

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  1. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Simon G. Sprecher

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Krieger, J., Spitzner, F. (2020). X-Ray Microscopy of the Larval Crustacean Brain. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 2047. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9732-9_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9732-9_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9731-2

  • Online ISBN: 978-1-4939-9732-9

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