The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4·0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS–PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

Vitellogenin (VTG) was isolated from the haemolymph of Penaeus vannamei by a three stepprocedure including ultracentrifugation, gel filtration and ion exchange chromatography. VTG wasused to raise polyclonal antibodies that were purified by ion exchange chromatography. A two stepcompetitive assay was developed in which VTG could be quantitated by its capacity to inhibit thebinding of antibody to the VTG previously adsorbed onto a solid phase. Sensitivity from equilibriumand from non-equilibrium assays was 41 and 2.3 ng/ml respectively. Estimates of within-assay andbetween-assay variabilities of standard curves were 4.2 and 9.6% respectively. The inhibition curves for dilutions of haemolymph from vitellogenic females, egg yolk extracts and purified vitellin (VTL) were parallel to the standard VTG curve, haemolymph from immature females and males showed no cross-reactivity. The antibodies directed against VTG recognize but partially VTL as shown by displacement curves. Recovery tests were near 100%. Thus the procedure was considered to be suitable for the measurement of haemolymphatic VTG. The VTG enzyme-linked immunosorbent assay (ELISA) developed in this study was validated by detecting physiological VTG changes in female shrimps after being fed squid extracts.

Isolation of vitellin from mature eggs of the palo verde beetle, Derobracus geminatus, was achieved by a combination of gel permeation and anion-exchange chromatography. It is a phosphoglycolipoprotein (Mr ˜250,000) that contains 8% lipids, 4% carbohydrates and 0.2% protein-bound phosphorus. The vitellin is composed of three apoproteins designated I (Mr ˜160,000), II (Mr ˜140,000) and III (Mr ˜50,000). The carbohydrate moiety is composed exclusively of mannose and N-acetylglucosamine. All three apoproteins are glycosylated as shown by flourescein isothiocyanate-conjugated concanavalin A and periodate-Schiff (PAS) staining suggesting the presence of a high mannose structure. Further evidence for the high mannose structure was obtained by the ability of endo-β-N-acetylglucosaminidase H treatment to remove the carbohydrate moieties on the apoproteins.

Two monoclonal antibodies (Mabs), that react in an enzyme linked immunoabsorbant assay, were generated against Aedes aegypti vitellin. One of these Mabs, VIIG9 that reacted predominantly with a 64 kDa component of Ae. aegypti yolk protein, also recognized 68 kDa and 66 kDa components in the yolk proteins of Culex sitiens and Cx. annulirostris, respectively. Mab VIIG9 has been used to detect vitellin or vitellin precursors in gravid female and non-blood fed nulliparous female mosquitoes as a possible age grading method. The presence of polyclonal and monoclonal antibodies to vitellin in the blood meal did not affect the fecundity of Ae. aegypti.