The silkrose of Bombyx mori effectively prevents vibriosis in penaeid prawns via the activation of innate immunity

Abstract

We previously identified novel bioactive polysaccharides from Bactrocera cucurbitae and Antheraea yamamai that activate innate immunity in RAW264 murine macrophages. However, in terms of potential applications in the cultivation of prawns, there were problems with the availability of these insects. However, we have now identified a polysaccharide from Bombyx mori that activates innate immunity in RAW264 cells and penaeid prawns. This purified polysaccharide, termed silkrose of B. mori (silkrose-BM), has a molecular weight of 1,150,000 and produces a single symmetrical peak on HPLC. Eight of nine constitutive monosaccharides of silkrose-BM are concomitant with dipterose of B. cucurbitae (dipterose-BC) and silkrose of A. yamamai (silkrose-AY). The major differences are found in the molar ratios of the monosaccharides. Silkrose-BM is approximately 500-fold less potent than silkrose-AY (EC₅₀: 2.5 and 0.0043 μg/mL, respectively) in a nitrite oxide (NO) production assay using RAW264 cells. However, the maximum NO production for silkrose-BM and AY were comparable and higher than that of the lipopolysaccharide of Escherichia coli. The survival of penaeid prawns (Litopenaeus vannamei and Marsupenaeus japonicus) after infection with Vibrio penaecida was significantly improved by both dietary silkrose-BM and B. mori pupae. This suggests that silkrose-BM effectively prevents vibriosis in penaeid prawns via the activation of innate immunity.

Figure 1

NO production activities stimulated by the addition of B. mori (circle) and A. yamamai (triangle) pupae to the culture medium of RAW264 cells.

Figure 2

Chromatogram of the purified polysaccharide of B. mori pupae on size-exclusive gel filtration chromatography.

Figure 3

Total ion chromatogram of the purified polysaccharide of B. mori pupae. 1, l-rhamnose; 2, l-fucose; 3, l-arabinose; 4, d-glucuronic acid; 5, d-mannose; 6, d-glucose; 7, d-galactose; 8, N-acetyl-d-glucosamine; 9, N-acetyl-d-galactosamine. Myo, Myo-inositol, an internal standard.

Figure 4

NO production activities in RAW264 cells stimulated by the polysaccharides purified from B. mori (circle) and A. yamamai (triangle) pupae. LPS of E. coli O26 was used as a positive control (cross).

Figure 5

Survival curves of L. vannamei after immersion with the Vibrio penaedia IAYKG13-1 strain (input dose: 3.8 × 10⁵ cells/L). Control diet group (filled circle), 0.0125 μg/g silkrose-BM diet group (open square), 0.250 μg/g silkrose-BM diet group (open triangle) and 5 μg/g silkrose-BM diet group (open circle). The cross indicates drop-out cases due to accidental death. Asterisks indicate statistically significant differences compared with the control group by log rank test with Bonferroni correction (p < 0.05). *p < 0.01, **p < 0.001, ***p < 0.0001.

Figure 6

Survival curves of M. japonicus after immersion with the Vibrio penaedia IAYKG13-1 strain (input dose: 3.6 × 10⁸ cells/L). Control diet group (filled circle), 0.001% B. mori pupae diet group (open square), 0.01% B. mori pupae diet group (open triangle), 0.1% B. mori pupae diet group (open circle). Asterisks indicate statistically significant differences compared with the control group by log rank test with Bonferroni correction (p < 0.05). *p < 0.01, **p < 0.001, ***p < 0.0001.