STUDY OF PROBIOTIC INHIBITION ON THE GROWTH OF Vibrio harveyi IN SHRIMP LARVAE USING MOLECULAR MARKER

Shrimp culture in Indonesia has become more intensive because of its high demand and economic value as export commodity. However, since 1990, shrimp culture has been suffered from many diseases problems, especially luminescent vibriosis caused by Vibrio harveyi.  Indeed, antibiotics can prevent a mass mortality, however, the wide spread antibiotic applications have also resulted some disadvantages including the emergence of antibiotic resistant pathogens and the possibility of antibiotic residual in the final products. Therefore, alternate eco-friendly method , i.e probiotic bacteria application could be proposed to solve this problem. Because of no Vibrio-free larvae available, the molecular marker is used as a reporter gene to analyze the disease inhibition by probiotic bacteria. Moreover, rifampicin-resistant marker can be used as molecular marker because almost all bacteria isolated from shrimp larvae and hatchery environments are sensitive to rifampicin. As molecular gene marker, gfp provides several advantages such as sensitive, stable, non-toxic, and visible, and can be detected in single cells. This study was carried out to observe the ability of probiotic bacteria to inhibit the growth of pathogenic V. harveyi in shrimp larvae using rifampicin-resistant and gfp molecular markers.

Probiotic bacteria used in this study was Vibrio alginolyticus SKT-b which marked by rifampicin-resistant marker (SKT-b^Rf-R). The pathogenic bacteria used in this study was V. harveyi G3 which marked by rifampicin-resistant marker (G3^Rf-R) and gfp (G3^gfp). The larvae were reared in three treatments; negative control (without administration of SKT-b^Rf-R and G3^Rf-R), positive control (with administration of G3 ^Rf-R or G3^gfp), and probiotic treatment (with administration of SKT-b^Rf-R and G3^Rf-R or G3^gfp). Parameters monitoring was done daily for 5 days including probiotic population, V. harveyi population, and survival rate of shrimp larvae.

The survival of larvae in negative control, positive control, and probiotic treatment were 85.0%, 36.7% and 75.0%, respectively. The administration of SKT-b into V. harveyi G3 treated larvae could increase the survival  of shrimp larvae. Recombinant V. harveyi harboring gfp (G3^gfp) showed green-fluorescent cells and could be observed inside the digestive tract of shrimp larvae.  The V. harveyi population in treated SKT-b probiotic shrimp larvae was lower than that without probiotic (Fig. 1), demonstrating possible competition for adherence sites or nutrition between V. harveyi and SKT-b.