Insect thromboxane mediates insect blood cell aggregation through calcium signal via a PG receptor

Innate immune responses are effective for insect survival under harsh environments to defend entomopathogens a fungal pathogen, Metarhizium rileyi, identified from field-collected larvae of a lepidopteran Spodoptera exigua. Although it exhibited significant pathogenicity against different insects, the virulence interfered with insect immune responses mediated by eicosanoids. Interestingly, the fungi induced Ca²⁺ signals in the hemocytes aggregating to the infection foci. The induction of Ca²⁺ signal was significantly inhibited by cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A₂ or B₂ (TXA₂ or TXB₂) is effective to recover Ca²⁺ signal and hemocyte aggregation. TXB₂ alone induced a micro aggregation behaviour under in vitro conditions. This is further supported by the induction of phospholipase A₂ (PLA₂) activity in the hemocytes to provide a biosynthetic precursor of the thromboxanes. Subsequently, expression levels of COX-like peroxynectins (SePOX-F and SePOX-H) were up-regulated upon the fungal infection. Finally, the expression of a thromboxane synthase (Se-TXAS) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited Ca²⁺ signal and hemocyte aggregation but hemocyte behaviour was rescued by the addition of TXB₂. Without any insect ortholog to mammalian thromboxane receptor, a previously known PG receptor was essential to mediate TXB₂ signal to mediate hemocyte aggregation behaviour. Altogether, these results suggest that hemocyte aggregation induced by fungal infection is triggered by TXB₂ via calcium signal through a PG receptor.