Vitellogenins appear in the form of protein complexes in the hemolymph of Haemaphysalis flava ticks

Abstract Background Hemolymph is a circulating aqueous fluid which is full of proteins, lipids, carbohydrates, hormones, and hemocytes. Ticks transport nutrients, signaling molecules, waste and immune factors to all areas of the body via hemolymph. Vitellogenin (Vg) family members, which are vital for the tick reproduction, are a main soluble component in the hemolymph, but there remain disputes regarding their protein structures and functional classifications. Here we aimed to investigate whether Vg proteins would exist in the hemolymph as a monomer or complex, as well as the putative functional classifications Vg proteins based on their structures. Methods Hemolymph was collected from fully engorged Haemaphysalis flava ticks by leg amputation. Hemolymph proteins were examined by both native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate PAGE (SDS-PAGE). Proteins extracted from gels were further identified by a combination of liquid chromatography–tandem mass spectrometry (LC–MS/MS). Results Two bands (380 kDa and 520 kDa) were separated from tick hemolymph by Native-PAGE and were further separated into four bands (105 kDa, 120 kDa, 130 kDa and 360 kDa) by SDS-PAGE. LC/MS/MS revealed that seven tick proteins and 13 host proteins existed in the four bands. These tick proteins mainly were the vitellogenin (Vg) family and α-macroglobulin family members. In silica structural analysis showed that these Vg family members all had common conserved domains including the N-terminus lipid binding domain (LPD-N), the C-terminus von Willebrand type D domain (vWD) and the unknown functional domain (DUF). Additionally, they contained the cleaving sites that could cleave the protein into multiple subunits. Meanwhile, combined with our previous observations, it was believed that these Vg family proteins in the hemolymph functionally belonged to carrier protein (CP) but not Vg. Conclusion These findings imply that Vg family proteins, most likely CP, are the primary constituent of Hemolymph in the form of protein complexes. It will drive a more rational selection of Vg family proteins as antigen targets for the development of vaccines aimed at the control of tick development.