A simple method for long-term vital-staining of ciliated epidermal cells in aquatic larvae


  • Jörn von Döhren Institute of Evolutionary Biology and Ecology, University of Bonn
  • Sabrina Kuhl Institute of Evolutionary Biology and Ecology, University of Bonn




cLSM, DiI, fluorescent staining, prototroch, Spiralia


Observing the process of growth and differentiation of tissues and organs is of crucial importance for the understanding of the evolution of organs in animals. Unfortunately, it is notoriously difficult to continuously monitor developmental processes due to the extended time they take. Long-term labeling of the tissues of interest represents a promising alternative to raise these pivotal data. In the case of the prototroch, a band of ciliated cells typical of marine, planktotrophic trochophora larvae, we were able to apply a long-term fluorescent vital-staining to the prototroch cells that remains detectable throughout further larval life. We were able to stain ciliated cells of planktonic larvae from different spiralian clades by using long-chain dialkylcarbocyanine dyes that are detectable in different fluorescent emission spectra in combination with a non-ionic surfactant. The larvae survived and developed normally, their ciliated cells retaining the originally applied fluorescent labels. Combined with additional fluorescent staining of the larvae after fixation, we provide an easy, versatile, and broadly applicable method to investigate the processes of the differentiation of epidermal organs in various aquatic larvae.

Author Biography

Jörn von Döhren, Institute of Evolutionary Biology and Ecology, University of Bonn

Dr. at the Institute of Evolutionary Biology and Ecology of the University of Bonn



Smart TI, von Dassow G (2009) Unusual development of the mitraria larva in the polychaete Owenia collaris. Biol Bull 217: 253–268.

Martín-Durán JM, Passamaneck YJ, Martindale MQ, Hejnol A (2017) The developmental basis for the recurrent evolution of deuterostomy and protostomy. Nat Ecol Evol 1: 5.

Hejnol A, Schnabel R (2006) What a couple of dimensions can do for you: comparative developmental studies using 4D microscopy – examples from tardigrade development. Integr Comp Biol 46: 151–161.

Lyons DC, Perry KJ, Henry JQ (2017) Morphogenesis along the animal-vegetal axis: fates of primary quartet micromere daughters in the gastropod Crepidula fornicata. BMC Evol Biol 17: 1–13.

Stach T, Winter J, Bouquet J-M, Chourrout D, Schnabel R (2008) Embryology of a planktonic tunicate reveals traces of sessility. Proc Natl Acad Sci 105: 7229–7234.

Raff RA (2008) Origins of the other metazoan body plans: the evolution of larval forms. Philos Trans R Soc B Biol Sci 363: 1473–1479.

Page LR (2009) Molluscan larvae: pelagic juveniles or slowly metamorphosing larvae? Biol Bull 216: 216–225.

Nielsen C (2019) Early animal evolution: a morphologist’s view. R Soc Open Sci 6: 190638.

Rouse GW (1999) Trochophore concepts: ciliary bands and the evolution of larvae in spiralian Metazoa. Biol J Linn Soc 66: 411–464.

Henry JQ, Hejnol A, Perry KJ, Martindale MQ (2007) Homology of ciliary bands in spiralian trochophores. Integr Comp Biol 47: 865–871.

Dorresteijn AWC (1990) Quantitative analysis of cellular differentiation during early embryogenesis of Platynereis dumerilii. Roux’s Arch Dev Biol 199: 14–30.

Schneider S, Fischer A, Dorresteijn AWC (1992) A morphometric comparison of dissimilar early development in sibling species of Platynereis (Annelida, Polychaeta). Roux’s Arch Dev Biol.;201: 243–256.

Kostyuchenko RP, Dondua AK (2006) Development of the prototroch in embryogenesis of Nereis virens (Polychaeta). Russ J Dev Biol 37: 69–76.

Damen P, Dictus WJAG (1994) Cell lineage of the prototroch of Patella vulgata (Gastropoda, Mollusca). Dev Biol 162: 364–383.

Tessmar-Raible K (2004) The evolution of sensory and neurosecretory cell types in bilaterian brains. Philipps-Universität Marburg. 210 p.

Borin M, Siffert W (1990) Stimulation by thrombin increases the cytosolic free Na+ concentration in human platelets. Studies with the novel fluorescent cytosolic Na+ indicator sodium-binding benzofuran isophthalate. J Biol Chem 265: 19543–19550.

Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671–675.

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona, A (2012) Fiji: an open-source platform for biological-image analysis. Nat Meth 9: 676–682.

Honig MG, Hume RI 1(986) Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures. J Cell Biol 103: 171–187.

Godement P, Vanselow J, Thanos S, Bonhoeffer F (1987) A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue. Development 101: 697–713.

Hofmann MH, Bleckmann H (1999) Effect of temperature and calcium on transneuronal diffusion of DiI in fixed brain preparations. J Neurosci Methods 88: 27–31.

Arai A, Nakamoto A, Shimizu T (2001) Specification of ectodermal teloblast lineages in embryos of the oligochaete annelid Tubifex: involvement of novel cell-cell interactions. Development 128: 1211–1219.

Henry JQ, Okusu A, Martindale MQ (2004) The cell lineage of the polyplacophoran, Chaetopleura apiculata: variation in the spiralian program and implications for molluscan evolution. Dev Biol 272: 145–160.

Willems M, Egger B, Wolff C, Mouton S, Houthoofd W, Fonderie P, Couvreur M, Artois T, Borgonie G (2009) Embryonic origins of hull cells in the flatworm Macrostomum lignano through cell lineage analysis: developmental and phylogenetic implications. Dev Genes Evol 219: 409–417.

Meyer NP, Boyle MJ, Martindale MQ, Seaver EC (2010) A comprehensive fate map by intracellular injection of identified blastomeres in the marine polychaete Capitella teleta. Evodevo 1: 1–27.

Gerberding M, Scholtz G (1999) Cell lineage of the midline cells in the amphipod crustacean Orchestia cavimana (Crustacea, Malacostraca) during formation and separation of the germ band. Dev Genes Evol 209: 91–102.

Kuwajima T, Sitko AA, Bhansali P, Jurgens C, Guido W, Mason C (2013) ClearT: a detergent-and solvent-free clearing method for neuronal and non-neuronal tissue. Development 140: 1364–1368.

Lukas JR, Aigner M, Denk M, Heinzl H, Burian M, Mayr R (1998) Carbocyanine postmortem neuronal tracing: influence of different parameters on tracing distance and combination with immunocytochemistry. J Histochem Cytochem 46: 901–910.

Wyeth RC, Croll RP (2011) Peripheral sensory cells in the cephalic sensory organs of Lymnaea stagnalis. J Comp Neurol 519: 1894–1913.




How to Cite

von Döhren J, Kuhl S. A simple method for long-term vital-staining of ciliated epidermal cells in aquatic larvae. J Biol Methods [Internet]. 2020Apr.29 [cited 2022Aug.11];7(2):e132. Available from: https://jbmethods.org/jbm/article/view/320