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 process of cellular immunity in insects and other animals that have open circulatory systems (Ratcliffe and Gagen, 1977). Efficient microorganism capture by nodules to prevent microbial infections has been reported in various insects, including Pieris brassicae, Galleria mellonella, Clitumnus extradentatus, Schistocerca gregaria, and Tenebrio moloitor (Ratcliffe and Rowley, 1979). Histologically, nodule formation has been described in two steps. The first involves granulocytes and oenocytoids (Ratcliffe and Gagen, 1977; Arai et al., 2013). Aggregates of hemocytes and microorganisms attach to the tissues within 1 min after microorganism injection (Ratcliffe and Gagen, 1977). In the silkworm, Bombyx mori, most of the nodules form within 30 s of Escherichia coli injection (Arai et al., 2013). Therefore, the first step of nodule formation plays an important role in the rapid capture of invading microorganisms. This first step was reported to be dependent on an adhesive protein called hemocytin (Arai et al., 2013), which is exocytosed by granulocytes (Ratcliffe and Gagen, 1977, Arai et al., 2013). Hemocytin present in the granules of granulocytes is released when microbial invasion is detected (Arai et al., 2013). However, few studies have investigated the role of hemocytes in this first step. In addition, it remains unclear whether nodule formation is a natural response or an unnatural one induced by injection of large amounts of microorganisms (1 × 106 cells). The second step occurs 2–6 h after microbial invasion and involves attachment of plasmatocytes to the core aggregates made in the first step to form a multicellular sheath (Ratcliffe and Rowley, 1979).

Initiation of the first step of nodule formation in B. mori is triggered through pathogen-associated molecular pattern recognition by C-type lectins present in the hemolymph, including B. mori lipopolysaccharide-binding protein and B. mori multibinding protein (Koizumi et al., 1997, Koizumi et al., 1999, Watanabe et al., 2006). Tokunaga et al. (2021) demonstrated that B. mori hemolymph proteinase 8 (BmHP8) is activated and binds to microorganisms within 15 min of incubation with hemolymph, whereas anti-BmHP8 antiserum strongly inhibits the first step of nodule formation following Saccharomyces cerevisiae injection (Tokunaga et al., 2021). By the way, the tobacco hornworm, Manduca sexta, has an extracellular signaling cascade that elicits the production of antimicrobial peptides (Kanost and Jiang, 2015). MsHP6 reportedly activates MsHP8, which in turn activates pro-spätzle1 (pro-SPZ1). Activated SPZ1 induces the production of antimicrobial peptides, probably through MsToll1 activation (An et al., 2009, 2010; Zhong et al., 2012). In Drosophila melanogaster, production of several antimicrobial peptides is induced by DmSPZ-mediated DmToll1 activation (Hoffmann, 2003). These molecules form an extracellular signaling pathway that produces antimicrobial peptides. Therefore, the extracellular signaling pathway involving BmHP8 is considered to mediate both antimicrobial peptide production and the first step of nodule formation.

Eicosanoids are C20 polyunsaturated fatty acids that mediate some insect immune responses (Stanley and Kim, 2014). In M. sexta and Spodoptera exigua larvae, nodule formation is strongly inhibited by dexamethasone, an intracellular eicosanoid biosynthesis inhibitor (Shrestha and Kim, 2009). In S. exigua, knockdown of SeMYD88 or SePelle, which are involved in the Toll signal transduction pathway, suppresses eicosanoid biosynthesis and nodule formation (Shafeeq et al., 2018). SeToll knockdown by RNA interference suppressed nodule formation but it was rescued using arachidonic acid, a putative eicosanoid biosynthesis intermediate (Park and Kim, 2012). Furthermore, knockout of S. exigua prostagrandin E2 receptor (SePGE2R) suppressed nodule formation (Kim et al., 2020). These observations, support a hypothesis that the SeToll signaling system activates some types of eicosanoid biosynthesis and induces nodule formation (Park and Kim, 2012). However, their data were evaluated 8 h after microorganism injection, so it is unclear which of the two nodule formation steps was induced by eicosanoids.

Serotonin (5-hydroxytryptamine; 5-HT) is a neurotransmitter and hormone biosynthesized from tryptophan. In humans, it has multiple physiological functions, including signal transmission in brain and thrombopoiesis. In S. exigua, 5-HT may be involved in nodule formation after E. coli injection (Kim et al., 2009). 5-HT antagonists suppress nodule formation, whereas 5-HT rescues it. Furthermore, 5-HT-induced nodule formation is inhibited by dexamethasone, an eicosanoid biosynthesis antagonist, suggesting that 5-HT acts downstream to eicosanoid release (Kim et al., 2009). This further suggests the possibility that the Toll signaling system may be a regulator of 5-HT synthesis instead of eicosanoid biosynthesis. However, Kim et al. (2009) evaluated 5-HT-induced nodule formation 10 h after microorganism injection, so it is unclear whether 5-HT secretion affects the first or the second step.

In the present study, approximately 400 hemocyte and S. cerevisiae cell aggregates appeared in the hemolymph within 1 min of S. cerevisiae cells injection (2 × 106 cells). We investigated whether these aggregates were precursors of nodules before adhering to tissues. We also investigated the role of these aggregates in microorganism elimination. Furthermore, we investigated whether 5-HT and eicosanoids, including prostaglandin E2 (PGE2), were relevant to aggregate formation using biosynthesis inhibitors and these compounds. We further investigated whether the aggregates generated by 5-HT and PGE2 were accompanied by hemocytin secretion. We found that hemocytes and microbial cells form aggregates within 1 min after the injection through microbial invasion-monitoring system in the hemolymph. We also demonstrated that nodule formation was not an unnatural response that required invasion of a large amount of microbial cells.

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