Light and ultramicroscopical aspects of the in vitro development of Hematodinium sp. (Dinoflagellata) from Atlantic snow crabs (Chionoecetes opilio)

Abstract

Hematodinium is a parasitic dinoflagellate of numerous crustacean species, including the economically important Atlantic snow crab, Chionoecetes opilio. The parasite was isolated from C. opilio in the fall of 2010 and 2011 and cultured in vitro. Several media were tested, with Nephrops medium yielding superior results. Life stages were characterized using light, scanning and transmission electron microscopy. This is the first time Hematodinium has been isolated from C. opilio and cultured in vitro, and is only the second in vitro study on Hematodinium which used electron microscopy. Numerous life stages were observed: amoeboid trophonts, sporonts, sheet-like and arachnoid coenocytes, dinospores, post-dinospores, and schizonts. In crab hemolymph, amoeboid trophonts and sporonts were observed. In cultures that were seeded with sporonts, further progression to motile dinospore stages occurred; however, those seeded with amoeboid trophonts did not progress to dinospores, often remaining as amoeboid trophonts or occasionally forming schizonts. Additionally, two types of coenocytes were associated with sporonts: sheet-like and arachnoid coenocytes. Only sheet-like coenocytes were associated with trophonts. Trophonts that were morphologically different were ultrastructurally similar, thus no distinction between filamentous and amoeboid trophonts was made. Ultrastructurally, trophonts possessed amphiesmal vesicle membranes, nuclei with permanently condensed chromosomes, mitochondria with tubular cristae, lipid droplets, and lipofuscin granules. Lipofuscin granules were characterized by their autofluorescence and ultrastructural similarity to lipofuscin granules found in other invertebrates and vertebrates. Additionally, lipofuscin granules appeared to degranulate or to be expelled from the cell during in vitro cultivation. The exact mechanism of this is unknown. Sporonts possessed all the features noted for trophonts and also had trichocysts. Mature trichocysts were long, rectangular shaped organelles with a width of 223 ± 2.11 nm (n=100) and of unknown length. Trichocyst development was examined ultrastructurally. Developing trichocysts were associated with the Golgi apparatus and first appeared as large vesicles containing a homogeneous, granular material. During development, the granular material condensed to form a crystalline core. Mature trichocysts were characterized by an electron dense crystalline core surrounded with a tightly juxtaposed single membrane. Early sporonts appeared similar to amoeboid trophonts in morphology; but became more spherical prior to dinospore formation. Late sporonts divided into four daughter cells, indicating a significant replicative step. Dinospores shared all ultrastructural features of sporonts, but possessed two flagella: one transverse and one longitudinal for motility. All dinospores were morphologically identical and resembled the macrodinospores observed in other Hematodinium spp. No microdinospores were observed; this lack of detection is likely due to the low number of observed cultures that progressed to the dinospore stage. After ~ 2 weeks in vitro, dinospores lost their flagella, and became more spherical. Furthermore, the nuclear structure was dissimilar to other stages, with beaded chromatin only evident along the periphery of the nucleus in addition to a single bead in the centre, rather than ubiquitous throughout the nucleus. Thus these were termed ‘post-dinospores’. Additionally, schizonts were noted as very large cells with large, relatively empty lipofuscin granules. Schizonts were only seen in contaminated or old cultures, and were thus considered as an abnormal form. Notably, no viable gorgonlocks or clump colonies were observed as noted in Hematodinium sp. from other crustacean hosts. In the present study, unique filopodia-like structures were observed protruding from early sporonts and trophonts and attached to neighbouring cells. Cryopreservation of Hematodinium was also attempted on sporonts and trophonts. Although recovery rates were very low, viable Hematodinium survived ~ 3 months in cryostorage. Two cryoprotectants, 3% glycerol and 10% DMSO, yielded positive results. This is the first successful cryopreservation of Hematodinium