Serotonin- and eicosanoid-dependent rapid hemocyte aggregation in the hemolymph is the first step in nodule formation in Bombyx mori larvae.

Nodule formation is a well-known process in cellular immunity of insects. However, few studies have investigated the role of hemocytes in rapid aggregation before tissue adhesion. In addition, since nodule formation is usually elicited by injecting large quantities of microbes, it remains unclear whether nodule formation is a natural response. The present study addressed these issues. A small number of nodules adhered to the dorsal vessels 1 min after Saccharomyces cerevisiae injection, while numerous aggregates of hemocytes and S. cerevisiae cells were observed in the hemolymph. The aggregate number decreased sharply after 5 min, corresponding to a rapid increase in the number of nodules. This suggests that aggregates formed in the hemolymph in response to S. cerevisiae injection eventually attached to the tissues. Nodules were induced using conditions that do not occur in nature, i.e., injection of nearly 2,000,000 S. cerevisiae cells. However, many aggregates contained only one S. cerevisiae cell, suggesting that aggregate formation can begin with the invasion of a single cell and that nodule formation is not an unnatural response. Biosynthesis inhibitors of serotonin (5-HT) and eicosanoids inhibited aggregate and nodule formation. In addition, injection of 5-HT and prostaglandin E2 induced hemocyte aggregation within 1 min in the hemolymph, along with hemocytin release. This suggested that 5-HT and eicosanoids induce rapid aggregation in response to invading microorganisms.

Nodule formation in Bombyx mori larvae is regulated by BmToll10-3.

Nodule formation is a two-step cell-mediated immune response that is elicited by the cytokine spätzle1. Spätzle1 is activated within 30 s of invasion by microorganisms via an extracellular signaling pathway that consists of pathogen-associated molecular pattern recognition receptors, C-type lectins, and serine proteases. Here, we investigated a hemocyte molecule that is involved in eliciting the first step of nodule formation. BmToll10-3 was one of 14 Toll homologs identified in the silkworm Bombyx mori; it is an ortholog of Spodoptera exigua Toll. Previous research suggested that SeToll elicits nodule formation, but no evidence was presented to indicate whether SeToll elicited the first or second step of nodule formation. Reverse transcription-polymerase chain reaction and immunostaining confirmed that BmToll10-3 is expressed in granulocytes. To determine whether BmToll10-3 is involved in eliciting the first step of nodule formation, we tested an antiserum raised against BmToll10-3 in a nodule formation assay. The antiserum strongly inhibited the first step of nodule formation in B. mori larvae. Next, we tried to knock out BmToll10-3 using genome editing. Strains that were heterozygous for a truncated BmToll10-3 allele were generated, but no strain that was homozygous for truncated BmToll10-3 was generated. Nonetheless, several healthy homozygous larvae were identified before pupation, and we used these larvae in a nodule formation assay. The larvae that were homozygous for truncated BmToll10-3 did not form nodules. These results suggest that BmToll10-3 is involved in a cellular immunity, nodule formation.