Maximum titers of vitellogenin and total hemolymph protein occur during the canalized phase of grasshopper egg production.

Many organisms exhibit developmental plasticity only in sensitive phases and cannot respond to environmental perturbations at other times. However, we know little about the physiological events that define plastic and canalized phases. During egg production in insects, vitellogenin (Vg) accumulates first in the hemolymph and then in the eggs. In addition, storage proteins may be important resources for egg production. Therefore, we tested hypotheses on the relationships of Vg and TP (total hemolymph protein minus Vg) titers to the transition from flexible to inflexible development during egg production. In lubber grasshoppers, approximately 70% of TP is contained in three proteins that range from 68 to 83 kDa. We maintained females on food treatments that produced defined plastic and canalized periods, collected hemolymph every approximately 4 d, and determined the ages at which oviposition and the maximum Vg and TP titers occurred. Both Vg(max) titer and especially TP(max) titer were predictors of the number of eggs produced. The time from eclosion to Vg(max) was significantly affected by diet, but the time from Vg(max) to oviposition was not. Similarly, the time from eclosion to TP(max) was significantly affected by diet, while the time from TP(max) to oviposition was not. Hence, Vg(max) and TP(max) are physiological landmarks that occur during the canalized phase of egg production.     

Juvenile hormone is a marker of the onset of reproductive canalization in lubber grasshoppers

To meet the challenge of unpredictable environments, many animals are initially developmentally flexible (plastic) but then may become inflexible (canalized) at major developmental events. The control of reproductive output can undergo a switch from flexible to inflexible (Moehrlin, G.S., Juliano, S.A., 1998. Plasticity of insect reproduction: testing models of flexible and fixed development in response to different growth rates. Oecologia 115, 492-500), and juvenile hormone (JH) may control this switch. By manipulating food availability, we tested the hypothesis that JH is involved in the reproductive canalization that appears during oogenesis in lubber grasshoppers. We used four food treatments: (1) high (H); (2) high switched to low (HL); (3) low switched to high (LH); and (4) low (L). We collected hemolymph samples approximately every 4 days and measured the ages at which maximum JH level (JH(max)) and oviposition occurred. Diet significantly affected both age at JH(max) and age at oviposition. In contrast, diet had no significant effect on the time from JH(max) to oviposition nor on the maximum JH level observed. Our data demonstrate that, after JH(max) is reached, the time to oviposition in our grasshoppers was unresponsive to food availability. Hence, reproductive timing appears to be canalized after the JH(max). This is the first demonstration in a phytophagous insect that a particular factor (in this case, JH) can be used to mark the switch from reproductive plasticity to reproductive canalization.     

Endocrine regulation of reproduction and diapause in the boll weevil,Anthonomus grandis Boheman

A review of the literature for the hormonal control of reproduction and diapause in Coleoptera is presented. The role of juvenile hormone and juvenile hormone esterase (JHE) in the control of the different life history strategies of the boll weevil are examined. Elevated levels of hemolymph JHE were found to be positively correlated with survival throughout the winter in South Texas population of weevils. Winter weevils were examined for hemolymph vitellogenin (Vg) and their subsequent survival was monitored. The majority of weevils surviving beyond ten weeks had no hemolymph Vg. We conclude that elevated hemolymph JHE and the absence of Vg are good predictors of survivors in the South Texas population. The hormonal basis of diapause termination was examined using hormone treatment and implant therapy. We were unsuccessful in our attempt to induce post-diapause reproductive development with all of our treatments. Only weevils given access to a food source were capable of reproductive development. Our recent experimental findings in the control of Vg synthesis and uptake in the boll weevil are reviewed. Arch. Insect Biochem. Physiol. 35:455–477, 1997. © 1997 Wiley-Liss, Inc.     

Characterization and quantification of yolk proteins in the lobster, Homarus americanus

Yolk protein (vitellin, Vn) and its precursor (vitellogenin, Vg) were isolated and characterized in the ovary and hemolymph, respectively, of the adult female lobster, Homarus americanus. Vn had a molecular mass of 360 kDa when analyzed by gel filtration. When analyzed by SDS-PAGE, Vn had six bands (110, 105, 94, 90, 81, and 78 kDa). An anti-Vn antiserum was developed using purified Vn, and the antiserum was used to detect Vn and Vg by ELISA and western blot techniques. ELISA analysis of hemolymph proteins separated by gel filtration indicated that Vg was similar in mass to Vn (360 kDa). However, western blots of hemolymph proteins separated by SDS-PAGE indicated that Vg contained a pair of protein subunits, 194 kDa and 179 kDa. Furthermore, the elution profiles of Vn and Vg from anion exchange chromatography indicated that Vg had a more negative charge. Thus, Vg appears to be processed after its uptake by the ovary to form Vn. Vg was undetectable in hemolymph from adult males by either ELISA or by western blot analysis. However, hemolymph levels of Vg in adult females increased 40-fold during the reproductive cycle, rising from 18 microg/mL in ovarian stage II to 789 microg/mL at stage V. This increase correlates well with oocyte growth during the cycle. Hence, this method may be useful for studying the regulation of lobster vitellogenesis.     

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.     

Quantification of vitellogenin-mRNA during maturation and breeding of a burying beetle

Burying beetles (Nicrophorus orbicollis) are unusual in that to breed they require an unpredictable and valuable resource, a small carcass. Thus the timing of reproduction is unpredictable and beetles' physiological response must be fast. We hypothesized that their pattern of vitellogenin (Vg) synthesis might reflect these requirements. We examined the expression of two Vg genes (sequenced for this study) during sexual maturation and through a reproductive bout. Vg-mRNA, juvenile hormone (JH) titers, ovarian development, and hemolymph concentrations of Vg were quantified in the same individuals. All four variables gradually increased during maturation to peak 15-20 days after eclosion. Twelve hours after the discovery of a carcass, a few hours before oviposition, mRNA was high, hemolymph Vg had decreased, JH and ovarian weight had increased. After oviposition, mRNA was low, hemolymph Vg concentrations and JH were high. This is consistent with our hypothesis that beetles produce and store Vg in the hemolymph prior to the discovery of a breeding resource and replace it quickly. Partial regression of these variables (with the effect of time removed) indicated that JH was not correlated with mRNA, hemolymph Vg, or ovarian weight at any time. Thus the role of JH as a gonadotropin remains unclear.     

Purification and characterization of biliverdin-binding vitellogenin from the hemolymph of the common cutworm,Spodoptera litura

Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage.     

Plasticity and canalization in the control of reproduction in the lubber grasshopper

The ability to change reproductive tactics during adult developmentin response to environmental variation is predicted to enhancefitness. Many organisms show phenotypic plasticity early innon-embryonic development, but later exhibit phases of developmentalinflexibility (=canalization). Therefore, we studied reproduction-relatedhormones and proteins and their relationships to plasticityin the Eastern lubber grasshopper. Diet-switching experimentsdemonstrated plasticity early in the egg production cycle, buta switch to canalization late in the cycle. We measured developmentaltiters of 4 hemolymph compounds from single individuals fromadult molt until first oviposition. These 4 compounds were theegg-yolk precursor protein vitellogenin, juvenile hormone (thecentral regulator of insect reproduction), major hemolymph proteins,and ecdysteroids (the arthropod molting hormone that ultimatelyis stored in the egg). Using diet manipulations, we investigatedhow these developmental titers relate to the switch from plasticto canalized egg production. All 4 hemolymph compounds reachedtheir peak levels during the canalized phase, about 12 day beforeoviposition. Diet switches after these peak levels did not affectthe timing to oviposition. Therefore, these peak titers werephysiological events that occurred after the individual committedto laying. We compared these patterns in reproduction to thedevelopment toward adult molt, another major life-history eventin insects. We observed an extended canalized phase before theadult molt. This canalized phase always included a peak of ecdysteroids.The similar patterns in the physiology of these life-historyevents suggested that common limitations may exist in majordevelopmental processes of insects that are directed by hormones.     

Vitellogenin synthesis,processing and hormonal regulation in the tick,Ornithodoros parkeri (Acari:Argasidae)

This study was undertaken to determine the processing of vitellogenin (Vg) and the role of juvenile hormone (JH) in the regulation of vitellogenesis in the tick Ornithodoros parkeri. Ticks usually require a blood meal to induce vitellogenesis. However, we have shown that a pyrethroid, cypermethrin (CyM), can stimulate Vg synthesis in unfed Ornithodoros moubata females. Vg concentration and synthesis were analyzed by SDS-PAGE spotting-scanning and fluorography using [³⁵S]-methionine. Although unfed females show high titers of Vg in the hemolymph, this is not due to new synthesis. Vg synthesis stimulated by engorgement increases beginning on day 2 after engorgement and reaches a maximum level on day 8. Vg is synthesized in the fat body, secreted into the hemolymph and then processed and incorporated into the ovaries as vitellin. JH I, II and III, methoprene (JHA), and CyM were topically applied to unfed females and Vg synthesis analyzed on day 5 by fluorography. JH and JHA did not stimulate Vg synthesis. CyM stimulated Vg synthesis but not ovarian development. These preliminary results indicate that JH does not function in the regulation of vitellogenin synthesis in this species.     

Vitellogenin and egg production in the moth,Heliothis virescens

The characteristics of vitellogenin (Vg) and the relationship between Vg production and egg production in the tobacco budworm, Heliothis virescens, were studied. The relationship between Vg production and juvenile hormone (JH) and the impact of mating on Vg and egg production were also investigated. Vg appears in the hemolymph of H. virescens about 6 h after moth eclosion. Vg may be separated into two apoproteins (ApoVg-I and ApoVg-II) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights were calculated to be 156,065 ± 800 for ApoVg-I and 39,887 ± 323 for ApoVg-II. SDS-PAGE analysis revealed that the female hemolymph Vg polypeptides appear to be identical to those from eggs but are absent in male hemolymph. Vg concentration was significantly higher in mated females than in virgin females of the same age at 48 h after emergence. Rates of egg production increased as Vg production increased; rates of egg production in mated females were significantly higher than those of virgin females at 48, 72, 96, and 120 h postemergence. Vg production is dependent on JH, because hemolymph from decapitated females lacked Vg while that of decapitated females treated with synthetic JH had Vg at levels comparable to similarly aged, normal H. virescens females. Hemolymph JH titers in mated females were significantly higher compared with those in virgin females at all sampling periods. The high JH level in mated females may explain the high Vg and egg production in mated H. virescens. Arch. Insect Biochem. Physiol. 34:287–300, 1997. © 1997 Wiley-Liss, Inc.     

JH III production, titers and degradation in relation to reproduction in male and female Anthonomus grandis

Juvenile hormone (JH) is necessary for the production of vitellogenin (Vg) in the boll weevil, Anthonomus grandis. Occurrence of Vg in this species is typically restricted to reproductively competent females, and is not detected in untreated males. However, the JH analog, methoprene stimulates Vg production in intact males and in the isolated abdomens of both male and female boll weevils (where in each case no Vg is detected without treatment), suggesting that males are competent to produce Vg but are normally not stimulated to do so. Preliminary work indicating that male boll weevil corpora allata (CA) produced little or no JH in vitro suggested that failure of males to produce Vg might be due to very low JH levels compared to females. This study re-examines the question of JH in male boll weevils by determining in vitro production of JH III by male CA during the first 10 days after adult emergence, determining hemolymph JH esterase activity during this same time period and hemolymph JH III titers in adults of both sexes. We also re-examine the ability of isolated male abdomens to produce Vg in response to hormonal stimulation, analyzing the effect of a wide range of methoprene and JH III dosages. Results indicate that male A. grandis have circulating JH titers and JH production similar to females. JH esterase activity is slightly but significantly higher in males than females. Vg production by isolated abdomens of both sexes after stimulation with methoprene or JH III was confirmed. Dose response studies indicated that high levels of methoprene were less effective than intermediate doses in stimulating Vg synthesis in both sexes. We conclude that the sexually dimorphic effect of JH on Vg synthesis is not due to differences in JH production or differences in JH titer between the sexes.     

Hemolymph ecdysteroids do not affect vitellogenesis in the lubber grasshopper

The role of hemolymph ecdysteroids in the reproduction of non-dipteran insects is unclear. We examine the role(s) of hemolymph ecdysteroids during egg production in the lubber grasshopper, Romalea microptera. In all individuals, hemolymph ecdysteroids rose to a sharp peak with similar maxima and then fell to undetectable levels. The time from the adult molt to the maximum ecdysteroid titer (E(max) titer) varied in response to food availability, whereas the time from E(max) titer to oviposition was unrelated to food availability. Because both the timing of egg production and the timing of E(max) responded similarly to environmental changes, ecdysteroids may be involved in egg production. We hypothesized that this role is the stimulation of vitellogenesis. Ovariectomized females had vitellogenin but no ecdysteroids, so ecdysteroids are not necessary for vitellogenin production. In addition, treatment of females with ecdysteroids altered neither Vg titers nor ovarian growth. Ovarian ecdysteriods increased at the same age in development as hemolymph ecdysteroids. In contrast to hemolymph ecdysteroids, ovarian ecdysteroids persisted until oviposition. Despite this, [(3)H]ecdysone injected into the hemolymph was detected later only at very low levels in the ovary, suggesting that hemolymph ecdysteroids are not sequestered by the ovary. In summary, our studies indicate that hemolymph ecdysteroids in adult females of the lubber grasshopper are associated with the timing of egg production, but they neither regulate vitellogenesis nor act as a source of ecdysteroids for the ovary.     

Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.     

Deduced primary structure of vitellogenin in the giant freshwater prawn, Macrobrachium rosenbergii, and yolk processing during ovarian maturation

A cDNA encoding vitellogenin (Vg) in the giant freshwater prawn, Macrobrachium rosenbergii, was cloned based on the cDNA sequence of vitellin (Vn) fragments A-N and B-42 determined previously, and its amino acid sequence deduced. The open reading frame (ORF) encoded 2,537 amino acid residues and its deduced amino acid sequence possessed three consensus cleavage sites, R-X-R-R, similar to those reported in Vgs of insects. The deduced primary structure of Vg in M. rosenbergii was seen to be similar to that of Penaeus japonicus, especially in the N-terminal region. It is therefore likely that Vgs in crustacean species including prawns and other related decapods exhibit a similar structural pattern. Based on the deduced primary structure of Vg and analysis of the various Vg and Vn subunits found in the hemolymph and ovary during ovarian maturation, we demonstrated the post-translational processing of Vg in M. rosenbergii. This is the first time that Vg processing has been clearly demonstrated in a crustacean species. Vg, after being synthesized in the hepatopancreas, is considered to be cleaved by a subtilisin-like endoprotease to form two subunits, A and proB, which are then released into the hemolymph. In the hemolymph, proB is possibly cleaved by a processing enzyme of unknown identity to give rise to subunits B and C/D. The three processed subunits A, B, and C/D are sequestered by the ovary to give rise to three yolk proteins, Macr-VnA, VnB, and VnC/D.     

Canalization of development and ecdysteroid timing during the last instar in lubber grasshoppers

Development in many phytophagous, holometabolous insects is flexible at the beginning but inflexible at the end of the last larval instar. A prominent feature of the inflexible period is a peak in hemolymph levels of ecdysteroids. We tested whether this pattern holds true for the final molt of a phytophagous, hemimetabolous insect, Romalea microptera (the Eastern lubber grasshopper). We fed one group of grasshoppers a high quantity diet (H) throughout the 5th (final) instar and a second group a low quantity diet (L) throughout the instar. Three other diet treatments involved starting the instar on the high diet and then abruptly switching to the low diet at 3, 8, or 13 days (H3L, H8L, and H13L respectively) and continuing the low diet until adult molt. Diet treatment did not affect the maximum hemolymph level of ecdysteroids (E(max)); this peak typically reached ~4000 ng/ml. Ecdysteroid levels were elevated for ~4 days in all groups. In contrast, diet significantly affected age at adult molt and age at E(max) such that H = H13L = H8L < H3L = L. We identified estimates of thresholds for weight gain (20% initial weight) and hemolymph ecdysteroids (100 ng/ml), after which diet did not affect the time to the adult molt. The weight gain threshold was less precise than the ecdysteroid threshold. These results suggest that R. microptera has an extended period of inflexible (canalized) development during the final instar that includes a peak of ecdysteroids. We hypothesize this pattern holds for many phytophagous, hemimetabolous insects.     

Hemolymph proteins in ticks

In comparison to insects and Crustacea, our knowledge of the predominant hemolymph proteins in ticks is minimal. The hemolymph protein most studied in ticks has been vitellogenin (Vg). Vg is synthesized by the tick fat body after female adults obtain a blood meal, is released into the hemolymph and is absorbed by developing oocytes as vitellin (Vn). Much of what we know about Vg is from studies of Vn. In general, the carbohydrate, lipid and amino acid composition is similar to insects except that in the tick, Vg contains heme, most likely from the digestion of host hemoglobin. In the American dog tick, Dermacentor variabilis, Vg is comprised of two native proteins and seven subunits on SDS-PAGE. Vg has been characterized in five tick species but the amino acid sequence is not yet available. Another predominant hemolymph protein, apparently a carrier protein (CP), has recently been studied in two tick species. This protein is found in the hemolymph of both male and females adults, in adult tissues outside of the hemolymph in some tick species, in coxal fluid of soft ticks and in whole body homogenates from eggs, larvae and nymphs. CP from the hard tick, D. variabilis, contains cholesterol, phospholipids, monoacylglycerides, triacylglycerides, free fatty acids, carbohydrate and heme. Under identical assay conditions, the analogous protein in the soft tick, Ornithodoros parkeri, did not contain heme. CP in the American dog tick consists of two subunits, one of which has 61% identity to the biliprotein, artemocyanin, from the fairy shrimp. CP is identical to a heme-lipoprotein (HeLp) from Boophilus microplus. The exact roles of CP and HeLp have not yet been fully determined, but they apparently are important in heme sequestration and as a storage depot for protein and lipid. Macroglobulin, lectin, antimicrobial, JH binding, JH esterase, and other tick hemolymph proteins are also discussed.     

Tebufenozide disrupts ovarian development and function in silkmoths

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.     

Effect of ecdysone agonists on vitellogenesis and the expression of EcR and USP in codling moth (Cydia pomonella)

The effects of tebufenozide and methoxyfenozide on vitellogenin (Vg) synthesis/release in the fat body, translocation in hemolymph, uptake by the ovary, and the expression of the ecdysone receptor (EcR) and its heterodimer partner, ultraspiracle protein (USP) in fat body, were investigated in Cydia pomonella. The results indicated that both ecdysone agonists significantly increased the Vg level in the adult hemolymph when the moths were exposed to agonist-treated surfaces. However, these agonists did not affect Vg release from the fat body nor Vg deposition in the first batch oocytes. Western blot analysis revealed that the expression of EcR and USP was significantly increased in tebufenozide- and methoxyfenozide-treated samples compared to the control, suggesting that ecdysone agonists regulated the Vg synthesis via the EcR and USP proteins complex.     

Vitellin of Pteromalus puparum (Hymenoptera: Pteromalidae), a pupal endoparasitoid of Pieris rapae (Lepidoptera: Pieridae): Biochemical characterization, temporal patterns of production and degradation

Vitellin (Vt) and vitellogenin (Vg) profiles were analyzed in Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. Non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses indicated that both native Vt and Vg were likely 370 kDa in size, consisting of two subunits of approximate 206 and 165 kDa. An indirect double antibody enzyme-linked immunosorbent assay (ELISA) for monitoring hemolymph Vg and ovarian Vt levels was developed using a monoclonal antibody and a polyclonal antibody made specially against P. puparum Vt. The synthesis and uptake of Vg in this wasp was initiated immediately after adult eclosion. The hemolymph Vg and ovarian Vt reached their highest level of 0.58 and 4.51 microg per female 24 and 48 h after adult eclosion, respectively. Both Vg synthesis and uptake were in parallel with ovarian development. The Vt levels in the developing embryos decreased progressively except 12h after parasitism. Meanwhile, nine new polypeptides with sizes ranging from 59.2 to 151 kDa, possibly resulting from the limited proteolysis of Vt originally accumulated in newly laid eggs, were detected de-novo during embryonic development using Western blotting with the monoclonal antibody against Vt. These studies provide the basis for future investigation into endocrinal regulations of vitellogenesis and understanding the reproductive strategy in this wasp.