Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment
Pathological Study of “Trembling Disease” in Chinese Mitten Crab Eriocheir sinensis
Zhifeng Gu, Wen Wang
Chinese mitten crab Eriocheir sinensis, an important aquatic product, is widely farmed in China. With its large-scale development, many crab diseases have become apparent. In recent years, the most serious one has been “trembling disease.” Once the disease occurs, it is difficult to control and causes high mortality. Therefore, many concerned parties have made great effort to study it; however, their conclusions vary. Lu et al. (1999), Chen et al. (1999), and He et al. (1999) found virus and virus particles; Yang and Cai (1998) thought its main pathogen was Vibrio; and Pan (1998) held that bacteria, parasite, virus, or another pathogenic microorganism was not the direct cause of the disease.
We have been studying the pathogen, pathogenesis, diagnosis, prevention, and cure of trembling disease in Chinese mitten crab since 1999. In this paper, the histology and ultrastructure of the disease are presented. We found a microorganism whose size is between that of virus and bacteria: rickettsia-like organism (RLO). After analyzing the form, invasion course, and characteristics of the microorganism, we concluded that RLO was the real pathogen.
Healthy crabs (70–100 g) were collected from a grow-out pond of East Mountain Lake, Su Zhou, in July 1999 and July 2000. They were cultured in 0.250-m3 aquariums equipped with a heating system, filter, and aerator and fed corn and spiral shells. The water was changed every week.
Sick crabs with the trembling disease trait (30–90 g) were collected at different times and from different ponds, including three lots from Hongze District in June 1999, one lot from Jinhu in June 2000, and three lots from Anhui in June 2000. The dying crabs were dissected and sampled immediately. Samples were fixed in glutaraldehyde, stored in an ice bottle, and transported to the laboratory.
The glutaraldehyde-fixed tissues of healthy and sick crabs were chopped fine, further fixed in osmium tetraoxid, embedded in Epon812, sectioned (semithin) with a Recherd–Jung ultramicrotome, and stained with methylene blue. The semithin sections were observed by using a light microscope and localized for ultrathin sections. The ultrathin sections were double stained with uranyl acetate and lead citrate, then observed and photographed by using a Hitachi H6000 transmission electron microscope.
A crab was wiped with 75% alcohol, and the fourth pair of pereiopods was removed. The flowing hemolymph was dropped onto a clean glass slide, smeared, flame dried, stained with Giemsa, dewatered with alcohol, and finally mounted with resin.
Early-stage sick crabs in ponds seldom eat or move; they lie at the bottom of water. After reaching the shore, they cannot return to the water because of pereiopod weakness. In the last stage of disease, sick crabs salivate and are unable to move or turn over once upside-down. When the crab is surprised, its pereiopods tremble fiercely.
The hemolymph of normal crabs was thicker than that of sick crabs and contained quite a lot of blood cells, whereas that of sick crabs had much fewer. Giemsa-positive particles were sometimes found in the cytoplasm of blood cells. Most of the blood cells had ruptured.
Ultrastructurally, RLOs were unexceptionally located in the connective tissue of appendage muscles, gill, midgut, hindgut, nerves, gonad, and other tissues of sick crabs; they were absent in the tissues of normal crabs. The RLOs (0.22–0.35 µm), were granular or clavate, each bounded by a cell wall and a membrane. In the center of the RLOs was no nucleus but a nuclear area, dark in color as viewed with transmission election microscopy, with a filiform structure around it (Figure 1).