Purification of vitellin and dynamics of vitellogenesis in the parthenogenetic tick Haemaphysalis longicornis (Acari: Ixodidae)

Abstract

Vitellin (Vt) was purified from eggs of parthenogenetic bush tick Haemaphysalis longicornis by gel filtration and ion exchange chromatography. Our results revealed that only one single Vt existed in parthenogenetic bush tick, and the purified Vt was proved to be a hemoglycolipoprotein consisting of nine polypeptides with molecular weights of 203, 147, 126, 82, 74, 70, 61, 47 and 31 kDa, respectively. Polyclonal antibody and monoclonal antibody against Vt were produced using the purified Vt. The change in vitellogenin (Vg) and Vt levels over time of the parthenogenetic H. longicornis was established, and the Vg content in haemolymph and Vt in ovary at different feeding or engorgement statuses was also determined using a double antibody sandwich enzyme-linked immunosorbent assay. The Vg level in haemolymph was distinctly increased on the day of engorgement (1.785 mg/mL) and continued to increase until 2nd day post-engorgement (5.611 mg/mL). There was a slight decrease in Vg level after 4 days of engorgement, and a second peak was observed on day 2 post-oviposition (10.774 mg/mL). Subsequently, Vg content continuously decreased and reached a low level on the 10th day post-oviposition. The Vt content in ovary continuously increased once the female reached its critical weight (0.024 mg per female), and reached the maximum level on day 2 post-oviposition (1.942 mg per female). Afterwards, Vt content rapidly decreased.

Ovary Proteome Analysis Reveals RH36 Regulates Reproduction via Vitellin Uptake Mediated by HSP70 Protein in Hard Ticks

“…Clathrin heavy chain involved in endocytic pathway regulates expression of vitellogenin in R. haemaphysaloides (Kuang et al, 2020). Vitellin is a hemoglycolipoprotein consisting of many bands (James and Oliver, 1997;Yang et al, 2015). Similarly, there were 11 polypeptides of vitellin identified by proteomic analysis in the R. haemaphysaloides ticks and vitellin antibody in this study recognized two bands of 136 and 72 kDa ( Supplementary Figure 4) that is different from the peptides of vitellogenin in ovary proteomics analysis (Supplementary Tables 4, 5), resulting in inconsistent phenomenon.…”

“…We speculated that RH36 was involved in the integration of nutrition and reproduction in ticks, in which vitellogenin is critical to tick ovary maturation since it serves as the nutritional source for tick survivability and reproduction (Xavier et al, 2018). Vitellogenin and vitellin are structurally, biochemically and immunologically similar in the majority of insects, including ticks, which results in McAb against vitellin recognized vitellogenins (Kaufman, 2004;Yang et al, 2015). Four vitellogenin genes ( Supplementary Figure 1) and McAb against vitellin (Figure 4) were obtained to test the dynamics of vitellogenesis.…”

“…Crude eggs were homogenized and subsequently centrifuged at 10,000 rpm for 20 min, the supernatant was applied onto a Sepharose CL-4B gel filtration column and diethyl-aminoethyl (DEAE)-cellulose column in turn (Yang et al, 2015). Polyclonal antibody and monoclonal antibody against vitellin was generated from Balb/c mice (Wang et al, 2017).…”

“…Splenocytes from immunized mice were fused to myeloma cells, SP2/0, using polyethylene glycol. Hybridomas were selected in HAT medium and the hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay (ELISA) in 96-microtiter plates and Western blot (Yang et al, 2015).…”

Investigation of three enzymes and their roles in the embryonic development of parthenogenetic Haemaphysalis longicornis

“…Although both cathepsin B and D were indicated to regulate embryogenesis, their dynamic changes were not the same in the parthenogenetic and bisexual populations. In our previous work, we found that the vitellin of the parthenogenetic population exhibits 9 subunits, ranging from 31 kDa to 203 kDa; however, in the bisexual population, vitellin exhibits only 8 subunits, ranging from 52 kDa to 112 kDa [44,45]. This might be another reason for the different dynamic changes in the enzymes, in addition to the longer incubation period.…”