Time course RT-qPCR study on immune, growth and stress-related gene expression in Bacillus subtilis supplemented overstocked shrimp “Litopeneaus vannmei”

The feasibility of Bacillus subtilis application on immune, growth, and stress responses in Litopeneaus vannmei has been assessed by evaluating changes over time of some immune, growth, and stress-related gene expression. A total of 315 L. vannamei weighted 0.78 g ± 0.08 (SE) divided into 3 experimental groups; the first group (T1) was considered as a control, the second group (T2) fed B. subtilis supplemented daily throughout the experiment. In contrast, the third group (T3) fed B. subtilis supplemented diet for a week followed by a week of basal diet alternatively. The expression levels of Toll receptor (LvToll), Penaeidin4 (PEN4), Protein kinase C delta type (PKC-delta), Ras-related protein Rap-2a (PAP-2a), Heat shock protein 70 (HSP70), and Heat shock protein 90 (HSP90) were evaluated before and after Vibrio  parahaemolyticus challenge. Results showed that down-regulation of LvToll, HSP70, and HSP90 expression levels was predominant in (T3) and transient up-regulation of PEN4, PKC, and RAP-2a levels expression in the same group in 1^st and 7^th week in which B. subtilis was supplemented. On the other hand, T2 showed up-regulation of lvToll and HSP70 in the 2^nd and 12^th weeks of trial, PEN4 in 1^st, 2^nd, and 7^th weeks, HSP90 in the 7^th and 12^th weeks; up-regulation of PKC and PAP-2a was observed nearly upon the entire experimental period. Immune response against V.  parahaemolyticus was on guard in (T3) than (T2). LvToll, PEN4, HSP70, HSP90, and PKC (T3) showed rapid upregulation in response to induced bacterial infection. RAP-2a responds to the bacterial challenge by significant down-regulation in both groups. This study suggests that the transient, weekly application of probiotic B. subtilis to shrimp may enhance the immune status, improve host stress tolerance, modulate pro-inflammatory responses, and trigger growth-associated responses of shrimp.