Application of bacterins and yeast Acremonium dyosporii to protect the larvae of Macrobrachium rosenbergii from vibriosis.

The fresh water prawn, Macrobrachium rosenbergii, has proven potential for use as an aquaculture species (Hanson & Goodwin, 1997; Kurup, 1984). In India alone, culture of this species of prawn in low saline areas requires about 200 million seed per year (Kurup, 1984). In hatcheries poor survival rate has been associated with vibriosis at different stages of the larval cycle. Members of the family Vibrionaceae associated with the larvae of M. rosenbergii were shown to be pathogenic under laboratory conditions (Bhat et al., 2000, in press). Vibrios have been associated with mortality of penaeid prawns by several workers (Aquacop, 1977; Hameed, 1993; Karunasagar et al., 1994). Two methods have been suggested to protect both the larvae and juveniles from vibriosis; one is the administration of bacterins prepared from pathogenic strains (Itami et al., 1989, 1991; Adams, 1991; Song & Sung, 1990; Sung et al., 1991) and the other is the utilization of yeast f 1-3 and 1-6 glucans as immunostimulants for enhancing the non-specific defense system (Sung et al., 1994; Song et al., 1997). In the light of these observations it was hypothesised that bacterins and yeast glucans may also be effective in protecting the larvae of M. rosenbergii from vibriosis as has been achieved in the case of penaeids. To examine this hypothesis, the ability of bacterins and an extracellular glue an-producing yeast to increase the overall survival and metamorphosis of larvae in a hatchery, as well as to protect against an experimental challenge under laboratory conditions, was evaluated. A strain each of Vibrio (ANM 708) and Photobacterium (AAC 727) isolated from diseased larvae of M. rosenbergii and found to be affiliated to V. fisheri, V. logei or V. marinus and P. angustrum, respectively, at G+C ratio level (determined from Tm values), but phenotypically dissimilar to the type strains and proven to be pathogenic to the larvae under laboratory conditions (Bhat et al., 2000, in press) were used for the preparation of bacterins. The bacteria were cultured in nutrient agar (Peptone 0.5%, Beef extract 0.5%) (HI Media Laboratories, Bombay, India), aged sea water (15 ppt), pH 7.5 at 28' C for 48 h and harvested in phosphate buffered saline (PBS) composed of NaH2PO4 6.42 g; Na2HPO4 34316 g; NaCl 10 g (SRL, Bombay, India), distilled water 1000 ml. The cultures were diluted in PBS to obtain 0.5 OD (Abs600) and were exposed to a final concentration of 0.2% (v/v) formalin for 24 h at room temperature and subsequently stored at 4' C for 14 days. To confirm inactivation an aliquot of 1 ml