Establishment of spiroplasma-infected hemocytes as an in vitro laboratory culture model of Chinese mitten crab Eriocheir sinensis.

Spiroplasma is a novel pathogen of commercially exploited crustaceans. To more full clarify its pathogenic mechanism at the molecular level in vitro, a spiroplasma-infected hemocytes model was developed as described here, using a modified method of hemocytes culture from the Chinese mitten crab Eriocheir sinensis. It has been standardized by employing L-15 growth medium supplemented with 15% fetal bovine serum along with 0.15% glucose, 0.75% NaCl, antibiotics (100 U ml(-1) penicillin, 100 U ml(-1) streptomycin) and a suitable pH of 7.20-7.40, incubated at 28°C without the requirement for 5% carbon dioxide. Cytopathic effects of Spiroplasma eriocheiris in the cultures, including cell debris and cellular exudates, were observed as early as 36 h post-inoculation. The green fluorescent dye Alex-488 was used as an immunofluorescence marker of S. eriocheiris to study its adhesion to, and infection of, the host cell. At about 24-h post-inoculation, a large number of spiroplasmas were observed infecting the host hemocytes cells, which became slug-like and widely distributed in the culture. Intracellular inclusion bodies containing spiroplasmas, and a large number of free spiroplasmas released from the ruptured cells, were observed using transmission electron microscopy. All the results show that S. eriocheiris can invade E. sinensis hemocytes in vitro, and this can provide a significant step forward toward further study of the relationship between the novel pathogen spiroplasma and its hosts in laboratory experimental studies.