Vitellin Research Articles

A Comparative Study on Vitellogenin Receptor of a Lepidopteran Insect (Spodoptera litura) and a Decapod Crustacean (Scylla serrata) : Phylogenetic Implication and Co-Evolution with Vitellogenins

Insects and crustaceans offer excellent model systems to study vitellogenesis in invertebrates. This paper gives a comprehensive account of the receptor-mediated yolk protein uptake in a lepidopteran insect Spodoptera litura and a decapod crustacean Scylla serrata . The vitellogenin (Vg) of both these arthropods has been characterized using electrophoretic and immuno-blotting techniques. The Vg's of both the animals are glycolipoproteins and exhibit immunological identity with their respective lipovitellins (Lv's). The physico-chemical characterization of vitellogenin receptors (VgR) of these arthropods revealed several similarities in their molecular weight and binding affinity with vitellins, which was increased considerably by divalent cation calcium. On the other hand, their binding affinity decreased significantly when treated with polyanionic suramin. These properties, along with the strong affinity of crab VgR for mammalian LDL and VLDL, qualify them as members of LDLR superfamily. The receptor-mediated endocytotic entry of Vg into the oocytes has been demonstrated by immunogold electron microscopic technique. The pathway starting from the formation of coated pits to the formation of mature endosomes has been traced using antibodies of Vg and VgR and gold-labeled secondary goat antibody. Use of green immunofluorescence techniques with FITC-tagged Vg antibody of S. litura produced additional evidence for endocytotic yolk protein uptake as well as uneven distribution of yolk within the oocytes.

Vaccine potential of a tick vitellin-degrading enzyme (VTDCE)

VTDCE (Vitelin-Degrading Cysteine Endopeptidase) is a peptidase with an active role in Rhipicephalus ( Boophilus) microplus embryogenesis. VTDCE is found in the tick's eggs and was shown to be the most active protein in vitellin (VT) hydrolysis of the three peptidases already characterized in R. microplus eggs ( Boophilus Yolk pro-cathepsin (BYC), Tick Heme Binding Aspartic Proteinase (THAP) and VTDCE). VTDCE activity was assessed in vitro using the natural substrate and a synthetic substrate (N-Cbz-Phe-Arg-MCA). The activity was inhibited by anti-VTDCE antibodies. In the present study, it was shown that VTDCE acts differently from BYC and THAP in VT hydrolysis and that the vaccination of bovines with VTDCE induces a partial protective immune response against R. microplus infestation. Immunized bovines challenged with R. microplus larvae presented an overall protection of 21%, and a reduction in the weight of fertile eggs of 17.6% was observed. The data obtained indicate that VTDCE seems to be important for tick physiology, and that it induces partial protective immune response when inoculated in bovines. This suggests that VTDCE can be useful to improve the protective capacity observed for other antigens.

Reproductive dysfunction induced by naphthalene in an estuarine crab Scylla serrata with reference to vitellogenesis

Biomarkers are useful tools for understanding complex interactions that elicit organisms response to environmental pollutants and their sublethal effects on organisms health. Effect of naphthalene on vitellogenin (VTG) and vitellin (VTN) were assessed in hepatopancreas, haemolymph and ovary of an estuarine crab Scylla serrata with reference to vitellogenic phases. In addition, the gonadosomatic index (GSI) was also assessed. Significant reductions in VTG and VTN contents were observed in hepatopancreas, haemolymph (VTG) and ovary (VTN). The GSI exhibited a decreasing trend in crabs exposed to naphthalene irrespective of the vitellogenic phases. We attempted to use the alterations in vitellogenic proteins and GSI as biomarkers of reproductive disturbances occurred in the crab due to naphthalene stress.

Kinetics of energy source utilization in Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) embryonic development

The present work evaluates the kinetics of utilization of the main potential energy sources throughout the embryonic developmental stages of Boophilus microplus. The embryonic development of this arthropod is completed in 21 days. Cellularization of the blastoderm occurs on the 6th day and is rapidly followed by germ band extension and segmentation, whose first signs are visible on the 7th day. Cellularization is typically a maternal-driven process, carried out by molecular determinants deposited in the oocyte during oogenesis. On the other hand, segmentation is of zygotic nature, being the consequence of the synthesis of various components by the growing embryo. The enhancement in total B. microplus RNA was observed after cellularization, corroborating the replacement of maternal-driven processes by embryonic zygotic expression. An abrupt increase in oxygen consumption was observed from cellularization until the 8th day of development. The reduction in dry weight at the same period and the susceptibility of oxygen consumption to KCN suggest that the respiration process is activated during early embryonic development. A marked decrease in total lipid content occurred between the 5th and 7th days of development, suggesting this is the main energy source for cellularization. A major reduction in carbohydrate content occurred later, between the 7th and 9th days, and it could be assigned to the morphological segmentation of the embryo. Although the total amount of proteins remains unchanged from oviposition to hatching, a 15% reduction in vitellin (VT) content was observed before cellularization, up to the 4th day after egglaying. This observation was correlated to the synthesis of new proteins needed to support early embryo development. Additional 20% of VT was consumed thereafter, mainly at the end of embryogenesis, and in this case VT is probably used as energy source to the older embryo. Altogether, these data indicate different energy sources for maternal and zygotic driven processes.

Tick vitellin is dephosphorylated by a protein tyrosine phosphatase during egg development: Effect of dephosphorylation on VT proteolysis

Vitellin (VT) is a phospholipoglycoprotein that is the main component of arthropod egg yolk. Phosphorylation is a recurrent feature of every VT molecule described so far. However, the role played by such post-translational modification during egg development is not yet clear. In the eggs of the hard tick Boophilus microplus, VT is a phosphotyrosine-containing protein. VT–phosphotyrosine residues are gradually removed during tick embryogenesis due to the action of a 45 kDa egg tyrosine phosphatase. This enzyme is strongly inhibited by ammonium molybdate, sodium vanadate and cupric ion. The role of phosphotyrosine residues in VT proteolytic degradation was evaluated. Western blots probed with a monoclonal anti-phosphotyrosine antibody demonstrated that the high molecular mass VT subunits (VT 1 and VT 2) are the main targets of dephosphorylation during egg development. Both dephosphorylation and proteolysis of VT 1 and VT 2 are blocked by ammonium molybdate in total egg homogenates. When purified VT was dephosphorylated in vitro with lambda phosphatase and then incubated in the presence of bovine cathepsin D, VT proteolysis increased dramatically. Altogether, these data are the first to show that phosphotyrosine residues are present in a yolk protein, and that such residues might be involved in the regulation of VT breakdown during egg development.

Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors

Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na +/K + tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods.

A vitellogenin chain containing a superoxide dismutase-like domain is the major component of yolk proteins in cladoceran crustacean Daphnia magna

A cDNA encoding vitellogenin (VTG), a precursor of a major yolk protein, vitellin (VTN), was isolated from cladoceran crustacean Daphnia magna. The deduced amino acid sequence of DmagVTG1, the polypeptide encoded by the cDNA, contained a possible signal peptide sequence of 16 amino acid (aa) residues. The possible mature form of DmagVTG1 consists of 1985 aa residues with a calculated molecular mass of 223,070 Da. The large lipid transfer (LLT) module and a part of the von Willebrand factor D (VWD) module found in the aa sequences of VTGs of many other organisms are well conserved in DmagVTG1. Phylogenetic analysis suggested that the LLT module of DmagVTG1 is more closely related to those of insect VTGs than those of decapodan crustaceans. A unique feature of DmagVTG1 is that it has a superoxide dismutase (SOD)-like domain at its NH 2-terminus. Antisera against the SOD-like domain, the NH 2-terminal part of the VTG domain and the COOH-terminal part of the VTG domain, respectively, were prepared and used for analysis of D. magna yolk proteins. Six species (I to VI) of major protein complexes were found in D. magna parthenogenetic eggs isolated immediately after ovulation. Complexes IV and V were the most abundant. DmagVTG1 was a component of Complexes III, IV and V, and the most abundant polypeptide in D. magna eggs. The protein complexes underwent gradual proteolysis during development. One of the primary sites of cleavage was between the two successive Arg residues located at the 1454th and 1455th positions of DmagVTG1.

Genetic parameter estimates for reproductive traits and egg composition in Pacific white shrimp Penaeus (Litopenaeus) vannamei

We estimated the heritabilities and genetic correlations for reproductive traits and egg composition in female shrimp, Penaeus (Litopenaeus) vannamei. The traits under analysis were days to first spawn after ablation (DTFS), egg diameter (ED) and number (EN), and egg triacylglycerides (TG), vitellin (VIT), total protein (PROT) and total lipids (LIP) for first spawned eggs. Estimates were based on 1–11 females from 30 full-sib families produced in 2000 and derived from the so-called ‘Melagos’ line in Mexico. Heritabilities were estimated using REML procedures from data on females that spawned at least once (56%). For ED, EN, and VIT, TG, PROT, and LIP in eggs, a square root transformation was done to normalize the data. Data was analysed using two models, one did not include any other trait as covariate, and the other included other trait as covariate. For DTFS the total number of spawns/female was used as covariate. For VIT, TG, PROT, and LIP in eggs the covariate used was egg number in that spawn. For egg number and egg diameter the other variable was reciprocally used as covariate. Heritabilities estimated without covariates were 0.41 for DTFS, 0.09 for EN, 0 for ED, 0.28 for VIT, 0.20 for TG, 0.13 for PROT, and 0 for LIP. Heritabilities for all traits but lipids increased when the covariates were introduced in the estimation of variance components; 0.54 for DTFS, 0.17 for EN, 0.07 for ED, 0.47 for VIT, and 0.35 for TG, 0.18 for PROT, and 0 for LIP. Despite large standard errors caused by small effective sample size, the estimates of the heritabilities indicate that these traits can be subjected to selection to improve reproductive output from females.

Proteolytic activity of Boophilus microplus Yolk pro-Cathepsin D (BYC) is coincident with cortical acidification during embryogenesis

In a previous report (Parasitology 116 (1998) 525) we isolated and characterized Boophilus Yolk pro-Cathepsin (BYC), an aspartic proteinase precursor from the eggs of the hard tick. The present study was designed to characterize the function of BYC in the consumption of vitellin (VT), the major yolk protein, during embryogenesis. Both purified BYC and total egg homogenate proteolytic activity showed a similar pH dependence profile with an acidic optimum. Purified BYC presented higher activity against VT as a substrate when compared to other proteins. The VT degradation pattern observed in vitro also showed a similar profile to that observed in vivo. Co-localization of BYC and acidic cortical yolk granules was performed by immunocytochemistry and confocal microscopy. Proton-pumping activity of yolk granules in vitro was higher in eggs collected 4 day after oviposition than in newly laid eggs. Taken together, our data suggest that BYC plays a major role in the degradation of VT and that its activity is controlled by acidification of yolk platelets localized at the cortical cytoplasm of the developing Boophilus microplus egg.

Relationship between vitellogenin and vitellin in a marine shrimp (Penaeus semisulcatus) and molecular characterization of vitellogenin complementary DNAs.

The source of yolk proteins in crustacean ovaries has been the subject of controversy for several decades, and both extraovarian and intraovarian synthesized proteins have been implicated. To offer a new insight into the relationship of vitellogenin (VTG) and vitellin (VT), a comparison of extraovarian VTG and ovarian VT of the marine shrimp Penaeus semisulcatus was performed at the protein and cDNA levels. Two cDNAs (7920 and 2068 nucleotides [nt]) were sequenced for VTG from the ovary and one cDNA (7920 nt) was sequenced from the hepatopancreas. VTG cDNA from hepatopancreas was similar to VTG cDNA from ovary. Although a VTG gene was also found in the males, approximately 7.8-kilobase transcripts were only detected in the ovary and hepatopancreas of females. The mRNA expression pattern was related to the stage of ovarian development and to the molt cycle, as determined by real-time polymerase chain reaction assay. VTG and VT apoproteins were composed of two and three major subunits, respectively, as shown by SDS-PAGE. N-terminal sequences of these subunits revealed the presence of a cleavage site at a consensus motif for a subtilisin-like endoprotease in VTG and VT and an additional cleavage site in VT revealed by an unidentified endoprotease. These results indicate that penaeid shrimps constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.

A Boophilus microplus vitellin-degrading cysteine endopeptidase.

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.

Response of Penaeus indicus females at two different stages of ovarian development to a lethal infection with Vibrio penaeicida.

An association between vitellogenesis and the immune system was suggested in crustaceans from studies on plasma lipoproteins. The present research studies the effect of an experimentally induced bacterial infection on vitellogenesis in females of the shrimp Penaeus indicus, as a model for penaeid species. Pre-vitellogenic and vitellogenic P. indicus females were experimentally infected with an extremely pathogenic bacterium, Vibrio penaeicida. The peak in mortality occurred earlier in pre-vitellogenic animals than in vitellogenic ones, although the final mortality level ( approximately 64-74%) 52h post-infection was nearly the same for the two groups. Twenty hours after infection, the total number of haemocytes was significantly reduced in vitellogenic females while there was no change in the pre-vitellogenic group. Protein synthesis in ovaries was not significantly affected by infection, at the two stages of ovarian development. No differences were found in mRNA levels of shrimp ovarian peritrophin protein (SOP), but preliminary results showed that mRNA expression of vitellin (VT) was reduced in a heavily infected vitellogenic female. The total amount of lipids in the haemolymph of vitellogenic females was almost twice higher than that of pre-vitellogenic ones. However, there was no change in the total content of lipids, lipid classes and fatty acid distribution in haemolymph or hepatopancreas following infection. Although vitellogenic and pre-vitellogenic females probably respond differently to a lethal bacterial infection, physiological differences may be concealed by the rapid onset of mortality.

In search of a vitellogenesis-inhibiting hormone from the eyestalks of the South African spiny lobster, Jasus lalandii

Summary Investigations into the control of reproduction in crustaceans have mostly concentrated on the regulation of female reproduction, specifically, the process of vitellogenesis which involves the accumulation of yolk proteins within the oocytes. The lipoglycoprotein, vitellin (VT), is a chief component of the yolk. In the current study, an attempt was made to identify a vitellogenesis-inhibiting hormone (VIH) from the sinus glands (SGs) of the spiny lobster, Jasus lalandii, by utilising a heterologous in vitro assay in which the incorporation of radiolabeled amino acids into proteins of shrimp (Penaeus semisulcatus) ovaries was assessed. Crude extracts of SGs, as well as purified neuropeptides from J. lalandii, were tested for VIH activity. We found that the crude extracts from the spiny lobster elicit a biological effect in penaeid shrimp ovaries at a physiological dose. Not all the purified eyestalk peptides inhibited de novo protein synthesis in the shrimp ovaries. VIH activity was not associated with a unique peptide but was ascribed to peptides with a hyperglycaemic effect in the spiny lobster. Moreover, all the ovarian proteins were affected by the active peptides. These results suggest that the term gonad-inhibiting hormones (GIH) is more appropriate for describing the biological function of cHH-family peptides than the term VIH. Furthermore, C-terminally shortened hyperglycaemic peptides did not have a significant effect on the inhibition of protein synthesis, indicating that the C-terminal portion of the cHH peptides is important for eliciting a biological function.

Cyclic nucleotide-independent phosphorylation of Vitellin by casein kinase II purified from Rhodnius prolixus oocytes

In this study we show that Vitellin (VT) phosphorylation in chorionated oocytes of Rhodnius prolixus is completely inhibited by heparin (10 μg/ml), a classical casein kinase II (CK II) inhibitor. VT phosphorylation is not affected by modulators of cyclic nucleotide-dependent protein kinases such as c-AMP (10 μM), H-8 (1 μM) and H-89 (0.1 μM). We have obtained a 3000-fold VT-free enriched preparation of CK II. Autophosphorylation of this enzyme preparation in the presence of 32P-ATP demonstrated that it lacks any endogenous substrates. Rhodnius CK II is strongly inhibited by heparin (Ki = 9 nM) and uses ATP (Km = 36 μM) or GTP (Km = 86 μM) as phosphate donors. Incubation of VT with purified Rhodnius CK II and 32P-ATP led to the incorporation of 2 mols of phosphate/mol VT. However, the total number of phosphorylation sites available can be altered by previous incubation of VT with alkaline phosphatase. These data show that an insect yolk protein contain phosphorylation sites for a cyclic nucleotide-independent protein kinase such as CK II.

Rhodnius prolixus vitellin is composed of three different populations: comparison with vitellogenin

Rhodnius prolixus oocyte extracts were chromatographed on an ion exchange column in order to purify vitellin (VT). Three VT heterogeneous populations were identified and named VT 1, VT 2, and VT 3 according to their order of elution from the column. The phosphate content of each population was determined, after lipid extraction, and a heterogeneous distribution was found: VT 1 being the less phosphorylated (50 mol P/mol protein) and VT 3 the heavily phosphorylated population (281 mol P/mol protein). Analysis of radioactivity associated with each VT population purified from animals fed with 32 Pi showed the same phosphorylation profile. Due to the fact that vitellogenin is the known precursor of VT, we have also chromatographed 32 P -VG in the same way as we purified VT. Only one VG's population was detected and resembled to VT 3 with respect to its elution profile. All VT populations contain the same neutral lipids, but they were heterogeneous with respect to phospholipid composition. VT 1 presents phosphatidylcholine and phosphatidylethanolamine whereas VT 2 and VT 3 also showed cardiolipin and probably phosphatidylserine. Sugar composition of VT 2 and VT 3 includes mannose as the main associated carbohydrate but VT 1 also contains glucose resembling VG. Although VG and VT are similar with respect to the elution profile, their sugar composition is different. These results suggest a post-endocytosis processing on VG molecule. The possible biological function of VT heterogeneous populations is discussed.

Characterization of vitellogenin in the red imported fire ant, Solenopsis invicta (Hymenoptera: Apocrita: Formicidae)

Vitellin (VN) and vitellogenin (VG) profiles were analyzed in monogyne and polygyne colonies of the red imported fire ant, Solenopsis invicta. Non-denaturing and SDS-polyacrylamide gel electrophoresis (PAGE) analyses indicated that the native VN was likely 350 kDa and comprised of two subunits in the molecular size range of 170–185 kDa. SDS–PAGE of hemolymph showed that the relative mobilities and subunit patterns of VG and VN were similar. VG was present in the hemolymph of reproductive queens; alate, virgin queens; and workers, but not in males. Anti-VN, prepared from polygyne egg homogenates, reacted with egg homogenates and with hemolymph VG from reproductive, monogyne and polygyne queens and alate, virgin polygyne queens. Analysis of circulating VG and ovarian development in alate, virgin queens showed that low levels of VG appeared by five days following adult eclosion, but egg development was not observed until seven weeks. VG was evident in newly inseminated queens, and increased steadily for the first three weeks following dealation. VG levels declined slightly near eclosion of the first workers (= nanitics) and dropped sharply after nanitic emergence at five weeks following dealation. Oocyte maturation peaked at days 15–25 following dealation, but otherwise remained low but steady. These studies provide the basis for future investigations into endocrine regulations of vitellogenesis in S. invicta queens.

A Heme-binding Aspartic Proteinase from the Eggs of the Hard TickBoophilus microplus

An aspartic proteinase that binds heme with a 1:1 stoichiometry was isolated and cloned from the eggs of the cattle tick Boophilus microplus. This proteinase, herein named THAP (tick heme-binding aspartic proteinase) showed pepstatin-sensitive hydrolytic activity against several peptide and protein substrates. Although hemoglobin was a good substrate for THAP, low proteolytic activity was observed against globin devoid of the heme prosthetic group. Hydrolysis of globin by THAP increased as increasing amounts of heme were added to globin, with maximum activation at a heme-to-globin 1:1 ratio. Further additions of heme to the reaction medium inhibited proteolysis, back to a level similar to that observed against globin alone. The addition of heme did not change THAP activity toward a synthetic peptide or against ribonuclease, a non-hemeprotein substrate. The major storage protein of tick eggs, vitellin (VT), the probable physiological substrate of THAP, is a hemeprotein. Hydrolysis of VT by THAP was also inhibited by the addition of heme to the incubation media. Taken together, our results suggest that THAP uses heme bound to VT as a docking site to increase specificity and regulate VT degradation according to heme availability.

Protein phosphorylation during Rhodnius prolixus embryogenesis: protein kinase casein kinase II activity

Protein kinase casein kinase II (CK II) activity was assayed during Rhodnius prolixus embryogenesis. Vitellin (VT) is the main endogenous substrate during the whole development. It is maximally phosphorylated at the third day of embryogenesis by CK II and then its phosphorylation decreases to a basal level by the time of first instar eclosion. When dephosphorylated casein was used as an exogenous substrate a different profile of enzyme activity was obtained. CK II activity increases on day 1 after fertilization and reaches a plateau on day 7 and its activity remains elevated until eclosion. Extracts obtained from oocytes or from 3-day old eggs were fractionated through gel filtration chromatography. CK II activity was assayed in each fraction and the enzyme obtained from the 3-day old eggs was shown to be three times more active than that obtained from oocytes, although the amount of enzyme present in the fractions was the same. These enriched CK II fractions were assayed against different effectors, such as: cAMP, H-8, H-89, calphostin C, sphingosine, polylysine and heparin. Heparin was the most effective one. When CK II activity was assayed in non-fertilized eggs, no activation of the enzyme was observed when compared to fertilized eggs. These data indicate that CK II is activated in a fertilization dependent process. The decrease in CK II activity against VT coincides with the beginning of VT proteolysis processing suggesting a possible relationship between protein phosphorylation and yolk degradation.

Vitellogenin uptake and vitellin localization in insect follicles examined using monoclonal antibodies and confocal scanning microscopy

Summary Confocal scanning immunofluorescent microscopy and monoclonal antibodies were used to examine the route of uptake of vitellogenin (VG) by vitellogenic follicles and the ooplasmic localization of vitellin (VN) in the cricket, Acheta domesticus, and the stick insect, Carausius morosus. Uptake and cytoplasmic regionalization of a non-vitellogenic sulfated protein, sp 157/85, by C. morosus oocytes were also examined. By indirect immunofluorescence VG in both species and sp 157/85 were visualized in spaces between follicle cells and in peripheral yolk spheres. One cricket VG polypeptide had a regionalized distribution in the folliclular epithelium, and VN polypeptides in both species and sp 157/85 in C. morosus had regionalized distributions within the ooplasm. Localization of sp 157/85 to the anterior pole of the oocyte appeared to be stage-specific.

Homologous ELISA procedure for the determination of penaeid shrimp vitellogenin

Resume Vitellogenin (VTG) was isolatcd from the haemolymph of Penaeus vannamei by a three step proccdurc including ultracentrifugation, gcl filtration and ion exchange chromatography. VTG was used to raise polyclonal antibodies that were purificd by ion exchange chromatography. A two step competitive assay was developed in which VTG could be quantitated by its capacity to inhibit the binding of antibody to the VTG previously adsorbed ont0 a solid phase. Sensitivity from equilibrium and from non-cquilibrium assays was 41 and 2.3 ng/ml respectively. Estimates of within-assay and between-assay variabilitics of standard curves were 4.2 and 9.6% respectively. The inhibition curves for dilutions of haemolymph from vitcllogcnic females, egg yolk extracts and purificd vitellin (VTL) were parallel to the standard VTG curve, haemolymph from immature females and males showcd no cross-reactivity. The antibodics dirccted against VTG recognizc but partially VTL as shown by displacemcnt curves. Recovery tests were ncar 100%. Thus the procedure was considered to be suitable for the measuremcnt oi 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.