Fig. 1: Serine protease inhibitor (SPI) I8 is conserved in nematodes of animals.

a Structures of SPI-I8 orthologs identified in blood-feeding Haemonchus contortus, Ancylostoma ceylanicum and Nippostrongylus brasiliensis and non-blood-feeding Heligmosomoides polygyrus are modelled and superimposed for similarity analysis. The sequences of SPI-I8 in nematodes were retrieved from WormBase ParaSite (WBPS18). The structures of the nematode SPI-I8s were modelled using the program AlphaFold2 (v. 2.1.0) and visualised with UCSF ChimeraX (v.1.0). Root-mean-square deviation (RMSD) scores are indicated. b Transcriptional profiling of serine protease inhibitor I8 coding gene (Hc-spi-i8) among the egg, first- (L1), second (L2), third- (L3), fourth-larval (L4), and adult stages of Haemonchus contortus. One-way variance analysis is used to indicate the significance of differential gene transcription among stages. Different letters indicate significance p < 0.05. c Using a polyclonal antibodies-based immunohistochemistry assay, SPI-I8 is localised to the inner membrane of hypodermis (indicated by white arrow) of female adult H. contortus. Mouse anti-Hc-SPI-I8A/ASA polyclonal antibody and pre-immunized mouse sera (control) were used as primer antibodies. Scar bars are 10 or 50 µm. DAPI 4’,6-diamidino- 2-phenylindole, FITC fluorescein 5-isothiocyanate, DIC Differential interference contrast microscopy. d Detection of SPI-I8 (isoform A/B) in the extracellular vesicles (EVs) and EVs-depleted excretory/secretory products (ESP*) released by the parasitic stages of H. contortus, using liquid chromatography–tandem mass spectrometry method. Number of proteins identified in EVs and/or ESP* is indicated.