Induction of antibacterial activity in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 in the anterior and posterior body part of the ligated larvae

Abstract

1. 1. Induction of antibacterial activity was investigated in the ligated fifth instar larvae of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 into the haemocoel in the anterior and in the posterior body part, followed by activity determination by inhibition zone assay of the haemolymph at 12 and 24 hr after immunization. 2. 2. At 12 hr after immunization, high antibacterial activity, approximately 6.8–7.5 mm in the anterior body part and 4.5–6.4 mm in the posterior body part in diameter of a clear zone (2.0 mm for no activity) was detectable in day 3 larvae. This result was in good contrast to expression of lectin gene in the ligated flesh fly, Sarcophaga peregrena (Shiraishi and Natori, 1988, FEBS Lett. 232, 163–166), in which only the anterior part of insect responded to stimulus of injury. 3. 3. Antibacterial activity at 24 hr after immunization in days 3 or 4 ligated larvae was lower than that at 12 hr; 4.0–4.5 mm of activity was observed in the anterior body part, and no activity was observed in day 3 ligated larvae in the posterior body part. 4. 4. Acid polyacrylamide gel electrophoresis of the haemolymph of immunized insects followed by overlay assay showed that the size of antibacterial activity bands were similar between the haemolymph from 12 hr and from 24 hr, and between the anterior and the posterior body part. This result was contradictory to the observation of activity by inhibition zone assay. The activity bands were associated with peptides that were similar to cecropin-like peptides A and B in the silkworm. 5. 5. These results indicate that in the silkworm, Bombyx mori, antibacterial peptides are induced both in the anterior and in the posterior body part of the ligated larvae. The results also suggest that some now unknown substance inhibiting antibacterial activity in inhibition zone assay appers preferentially in the posterior body part of the ligated larvae at 24 hr after immunization.