|
without Mg and Ca(EBSS) in 96-well plate, one per-well, and were incubated at room temperature. The development stages of trophonts were monitored and observed under inverted microscope and image analysis system.
Results & Discussion
The trophonts in 96-well plate encysted in all the three media as well as the sterile filtered seawater within 24 hours (Plate 1). The results of the survival of C. irritans from different media are showed in Table 1. No dead trophonts were found on day one in seawater, HBSS and MEME except in EBSS. On day 2, there were signs of cell division inside the thick-walled cyst and from day 5-7, moving tomites were observed in sterile seawater and HBSS(Plate 2). In the EBSS medium cell division was observed from day 3 up to day 6, but no tomites was produced. In contrast, no cell division of the trophont, which remain encysted up to day 9 in MEME media through out the experimental period.
The life cycle of C. irritans involves four developmental stages namely as trophont(grows within the epidermis of the skin and gills of fish), tomont encystment (a trophont stage that leaves the host and secretes a cyst or a thick wall surround the body surface), division tomont (an encystment trophont undergoing cell divisions to form tomites), and theronts(individual cell in division tomont). Tomonts are characterised by a wall composed of several layers, and also by their unequal cell divisions. According to Colorni and Diamant (1993), they divide and develop into theronts in an asynchronous rate. The number of tomonts completing encystment, threonts produced per cyst and theronts subsequently surviving were found to be different in all the media tested. When the trophont was placed in the HBSS, EBSS and MEME media, it encysted on the first day. However, the encysted trophont in MEME medium did not show any sign of cell division, whereas it showed cells division in seawater, HBSS and EBSS media (Figure 1).
Reproduction in C. irritans is generally associated with the production of tomites during encystment, which differentiate into theronts. However, the detection of precystic trophont division here in culture may be indicative of a normal, although rarely observed event in the life cycle of this parasite. In the present study, it is suggested that reproduction may occur within the fish epidermis as recorded a single precystic division of trophonts following exit from the host epidermis and the subsequent normal encystment and development of the progeny. The mature trophont leaves the fish as a free-swimming tomont, secretes a cyst and undergoes multiple divisions to produce tomites. Tomites emerge as infective free-swimming theronts that attach and penetrate into the skin and gill epithelia of susceptible fishes. The secretion of the cyst is usually completed within 24 hours in 25oC (Dickerson and Dawe, 1995).
Multiple divisions by budding were also recorded in culture, resulting in numerous small individuals, probably representing tomite production. Because of its thickness and hardness, the cyst wall of the tomont retains its original shape after swimming out of the tomites. According to Cheung et al. (1979), successful reproduction in vitro requires the secretion of a cyst
|
|
