Modulation of Silkworm Haemolymph Proteins and Proteases by Chitosan: A Defense Mechanism against Bacterial Infection

Flacherie, a contagious bacterial disease caused by Staphylococcus aureus and Bacillus thuringiensis, presents a critical constraint in tropical sericulture. To probe host-pathogen biochemical interactions, this study investigated total protein content and protease enzyme activity in haemolymph of Bombyx mori (bivoltine hybrid FC1 × FC2) following oral bacterial inoculation and treatment with silkworm pupal chitosan. Silkworm rearing adhered to standard protocols. Pupae-derived chitosan was extracted via sequential deproteinization, demineralization, deacetylation and decolorization. Concentrations of 3500-5000 ppm extracted chitosan as well as 1000 ppm commercial chitosan and 1000 ppm ampicillin were applied to mulberry feed at 6, 12, 18, 24 and 30 hours post-bacterial inoculation (hpi). Haemolymph samples were collected daily from the second to the eighth day of the fifth instar for biochemical analysis. Results demonstrated that total protein content varied significantly by bacterial strain, treatment type, timing and their interactions. Chitosan treatments (3500-5000 ppm) and ampicillin significantly raised haemolymph protein levels over days 1-8, in contrast to control groups, which declined initially and rose only modestly later. Protein levels were consistently higher in S. aureus infections compared to B. thuringiensis. Early chitosan application produced better outcomes, while delayed treatment corresponded to reduced protein accumulation. Protease activity exhibited a similar pattern: enzyme activity increased from day 2 to day 8 across all treatments, peaking in uninfected controls, followed by ampicillin, commercial chitosan and 5000 ppm extracted chitosan treatments. Lowest protease activity occurred in absolute controls. Again, S. aureus induced higher protease activity than B. thuringiensis and delayed chitosan addition led to attenuated enzymatic response. This study reveals that early application of high‑concentration silkworm‑pupal chitosan significantly enhances immune‑related protein content and protease activity, especially under S. aureus infection. These findings underscore chitosan’s immuno-stimulatory potential in sericulture and encourage further mechanistic studies to optimize its use as a natural disease‑management strategy in silkworm rearing.