Molecular and cellular analyses of a ryanodine receptor from hemocytes of Pieris rapae

Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are a distinct class of ligand-gated calcium channels controlling the release of calcium from intracellular stores. Intracellular calcium level has a definite role in innate and adaptive immune signaling. However, very few information are accessible about calcium transients of invertebrate immunocytes, especially of insect hemocytes, the effector cells of insect immunity. In this study, we show that the RyR-stimulating agent flubendiamide inhibit hemocyte spreading and phagocytosis in the cabbage white butterfly, Pieris rapae. Furthermore, we cloned a cDNA encoding a ryanodine receptor (PrRyR) from the hemocytes of P. rapae. It encodes 5107 amino acids with a predicted molecular weight of 578.2 kDa. PrRyR shares a common feature with known RyRs: a well-conserved COOH-terminal domain with two consensus calcium-binding EF-hands and six transmembrane domains, and a large hydrophilic NH2-terminal domain. In the larval stage, PrRyR was highly expressed in epidermis tissue and also expressed in hemocytes at a moderate level. In the adult stage, PrRyR was expressed at high levels in thoraces and legs, while low levels in abdomens and antennae. Quantitative real-time PCR analysis showed that its expression did not display any significant change in response to bacterial challenge. Western blot analysis and immunohistochemistry assay displayed that PrRyR was detected and presented on hemocytes. We also showed that flubendiamide, a RyR-activating insecticide, induced Ca(2+) release and thereby confirmed functional expression of the PrRyR in the hemocytes of P. rapae.