AbstractCotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae), a solitary braconid endoparasitoid wasp, parasitizes the diamondback moth Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) by suppressing the host defense response, thereby resulting in successful parasitization. During parasitization, ovarian calyx fluid is also delivered into the haemocoel of the host along with the wasp egg. The effect of calyx fluid constituents on haemocyte-spreading behaviour of P. xylostella is analysed by measuring F-actin development in the haemocytes. For this purpose, the calyx fluid of C. plutellae is separated into ovarian protein and C. plutellae bracovirus (CpBV). The ovarian protein consists of a wide range of molecular weight proteins, which are apparently different from those of CpBV. When nonparasitized P. xylostella haemocytes are incubated with either ovarian protein or CpBV for 1 or 2 h, haemocytes lose their responsiveness to a cytokine, plasmatocyte-spreading peptide, in a dose-dependent manner for each calyx component and fail to exhibit haemocyte-spreading behaviour. Some CpBV genes are expressed within 1 h of parasitization. The inhibition of haemocyte-spreading could be explained by measuring F-actin contents, in which parasitization by C. plutellae inhibits F-actin development in the haemocytes of P. xylostella. Either ovarian protein or CpBV could inhibit F-actin development in the nonparasitized haemocytes. In addition, co-incubation of ovarian protein and CpBV results in significant additive inhibition of both haemocyte-spreading and F-actin development in the haemocytes in response to cytokine. These results suggest that both components of C. plutellae calyx fluid function in a synergistic manner, leading to immunosuppression during the early stage of parasitization.