Ultrastructural and functional characterization of circulating hemocytes from the freshwater crayfish Astacus leptodactylus: Cell types and their role after in vivo artificial non-self challenge Piero Giulio Giulianini * , Manuel Bierti, Simonetta Lorenzon 1 , Silvia Battistella, Enrico Antonio Ferrero Department of Biology, University of Trieste, via Licio Giorgieri 7, I-34127 Trieste, Italy Received 20 January 2006; received in revised form 30 March 2006; accepted 30 March 2006 Abstract The freshwater crayfish Astacus leptodactylus (Eschscholtz, 1823) is an important aquacultured decapod species as well as an invasive species in some European countries. In the current investigation we characterized the different classes of circulating blood cells in A. leptodactylus by means of light and electron microscopy analysis and we explored their reaction to different latex beads particles in vivo by total and differential cell counts at 0.5, 1, 2 and 4 h after injections. We identified hemocytes by granule size morphometry as hyaline hemocytes with no or rare tiny granules, small granule hemocytes, unimodal medium diameter granule hemocytes and both small and large granule containing hemocytes. The latter granular hemocytes showed the strongest phenoloxidase L-DOPA reactivity both in granules and cytoplasm. A. leptodactylus respond to foreign particles with strong cellular immune responses. All treatments elicited a total hemocyte increase with a conspicuous recruitment of large granule containing hemocytes. All hemocyte types mounted some phagocytic response but the small granule hemocytes were the only ones involved in phagocytic response to all foreign particles with the highest percentages. These results (1) depict the variability in decapod hemocyte functional morphology; (2) identify the small granule hemocyte as the major phagocytic cell; (3) suggest that the rather rapid recruitment of large granule hemocyte in all treatments plays a relevant role by this hemocyte type in defense against foreign particles, probably in nodule formation. # 2006 Elsevier Ltd. All rights reserved. Keywords: Hemocytes; Ultrastructure; Latex beads; Phagocytosis; Cellular immunity; Astacus leptodactylus (Crustacea) 1. Introduction The circulating hemocytes of crustaceans play a central role in innate immunity. They are involved in nodule formation, encapsulation and in phagocytosis. Three hemocyte types are commonly described in crustaceans: hyaline hemocytes (agranular), small granule (semigranular) hemocytes, and large granule (granular) hemocytes (Mix and Sparks, 1980; Bauchau, 1981; So ¨derha ¨ll and Smith, 1983; Martin and Graves, 1985; Hose et al., 1990; Johansson et al., 2000; Battison et al., 2003). The morphological classes are essentially based on granule number and size and on nucleus to cytoplasm ratio by using phase contrast or brightfield light microscopy and transmission electron microscopy. Martin and Graves (1985) pointed out that routine staining procedures of hemolymph smears are not useful because they are often ambiguous, not easily duplicated and the cell morphology is highly distorted. Recently, drawbacks induced by different light microscopy techniques on live cells and staining of unfixed hemolymph smears have been overpassed by authors that used cytocentrifugation of diluted fixed hemolymph followed by Wright-Giemsa staining and obtaining permanent stained slides (Horney et al., 2002; Battison et al., 2003). The latter technique allowed discrimina- tion of 11 hemocyte types, enabling to observe less mature hemocytes in late stages of Aerococcus viridans var. homari infection (Battison et al., 2003). The number of circulating hemocytes is also important and it can decrease dramatically after bacterial infection or following various types of stresses (Lorenzon et al., 2001, 2002). The freshwater crayfish Astacus leptodactylus (Eschscholtz, 1823) is an economically important aquacultured species as well as an invasive species in some European countries. The cellular immune response of A. leptodactylus has gone www.elsevier.com/locate/micron Micron 38 (2007) 49–57 * Corresponding author. Tel.: +39 040 5583560; fax: +39 040 575079. E-mail address: giuliani@units.it (P.G. Giulianini). 1 Present address: Osservatorio Geofisico Sperimentale, Dipartimento di Oceanografia Biologica, via Picard, 54, I-34010 Trieste, Italy. 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.03.019