Synthesis and secretion of a precursor hemolymph juvenile hormone-binding protein in the adult female cockroach Leucophaea maderae

Hemolymph juvenile hormone-binding protein (JHBP) is synthesized and secreted from fat body in the adult female cockroach, Leucophaea maderae. The data in this paper suggest it is initially secreted from the fat body as a larger peptide whereas data in the accompanying paper demonstrate that JHBP is apolipophorin I. Using media from cultures of fat body maintained in vitro, a JH-binding component was found that is JH III saturable, has a K_D of 1.5 × 10⁻⁸ M, binds JH III > JH II > JH I, and has a sedimentation value of 6.5S on high salt sucrose gradients. Each of these properties is identical to those of the JHBP extracted from the hemolymph. To identify the protein that bound JH, media proteins were photoaffinity labeled with 10-[10,11-³H]epoxyfarnesyl diazoacetate ([³H]EFDA). The results revealed that two media proteins bound [³H]EFDA in the absence of JH III, but not in the presence of 100-fold excess JH III. The molecular weights of the two media peptides were estimated by SDS-PAGE to be 275,000 and 220,000.To determine if the JHBP found in media of fat body cultures was due to hemolymph contamination of fat body, incorporation of [³H]leucine into newly synthesized and secreted fat body proteins during a 48 h culture period was monitored. During the culture period, linear increases in the concentrations of radiolabeled 275 and 220 kD JHBP were observed. Monoclonal antibodies specific for the 220 kD hemolymph JHBP were found to recognize both the 275 and 220 kD JHBPs in the media.To investigate the possibility that the 275 kD protein is a precursor to the 220 kD protein and that components of the hemolymph process or modify the precursor, hemolymph was introduced into fat body cultures and relative concentrations of the 275 and 220 kD media JHBPs were determined. Addition of hemolymph to these organ cultures resulted in an increase in the concentration of radiolabeled 220 kD JHBP and a proportional decrease in the concentration of radiolabeled 275 kD JHBP, suggesting that the 275 kD protein is a precursor to the 220 kD hemolymph JHBP. The mechanism of processing or modification remains undetermined.     

Rapid purification and N-terminal amino acid sequence of a photoaffinity-labeled juvenile hormone binding protein from an arctiid moth larva,Platyprepia virginalis

A novel two-step procedure has been developed for the purification of juvenile hormone binding proteins (JHBP) from caterpillars. Crude hemolymph was photoaffinity labeled with [³H]EHDA, a JH II analog. After removal of excess ligand, 40 ml of buffer-diluted hemolymph containing over 200 mg protein was submitted to preparative isoelectric focusing (IEF) using a Rotofor device. After removal of ampholytes by dialysis, the ³H-labeled fractions were purified to > 95% homogeneity by anion-exchange HPLC. Over 1000-fold purification could be achieved in a few days on a scale which provides 100–1000 μg of purified JHBP. Proteins thus obtained can be used for proteolytic digestion or can be sequenced after electroblotting from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel onto a polyvinylidene fluoride (PVDF) membrane. This protocol is illustrated for the purification and N-terminal amino acid sequencing of a hemolymph JHBP from an arctiid wooly bear caterpillar, Platyprepia virginalis.     

Synthesis and release of lipophorin in larvae of the southwestern corn borer,Diatraea grandiosella: An in vitro study

The source of the lipophorin present in the larval haemolymph of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Although lipophorin was shown to be one of several proteins released from cultured fat body and midgut, only fat body was shown to synthesize lipophorin. Fat body, incubated in a medium containing [³H]leucine, was shown to release radiolabelled lipophorin using immunoprecipitation. Similar studies using midguts incubated in a medium containing [³H]leucine did not reveal any synthesis of lipophorin. Lipophorin was isolated by density-gradient ultracentrifugation from media in which the fat bodies of about 600 diapausing larvae had been incubated for 4 hr. The isolated lipophorin had a peak density of 1.11 g/ml, and contained various lipids including diacylglycerol, triacylglycerol, sterol, hydrocarbon, free fatty acid, phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin.     

Purification of the Drosophila Kc cell juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) from the cytosol of Drosophila melanogaster Kc cells has been purified with the use of a juvenile hormone photoaffinity analog, 10,11-epoxy (2E, 6E) farnesyl diazoacetate (EFDA). The purification procedure consists of five chromatographic steps and the end product of the purification procedure showed homogeneity by means of both native and SDS polyacrylamide gel electrophoresis. Furthermore, using a racemic mixture of the natural hormone, [³H]juvenile hormone III (JH III), as the radioligand in this purification procedure, we demonstrate that the purified protein is likely the authentic intracellular JHBP.     

In vitro synthesis and secretion of lipophorin by the fat body of nondiapause and prediapause larvae of the southwestern corn borer,Diatraea grandiosella

The capacity of the fat body of nondiapause, prediapause and diapause larvae of the southwestern corn borer, Diatraea gradiosella, to synthesize and release lipophorin was examined in vitro using [³H]leucine as the radiotracer. Synthesis and release of [³H]lipophorin by the fat body peaked in 11–13 day-old fifth instar nondiapause larvae, which coincided with their feeding period. The rate of lipophorin synthesis in the fat body of newly ecdysed pupae was extremely low. Synthesis and release of [³H]lipophorin by the fat body of prediapause larvae occurred at the highest rates in 20–35 day-old fifth and sixth instars, and declined to virtually undetectable levels after larvae entered diapause around 40 days-of-age. Immunoprecipitation of [³H]lipophorin from fat body of 13 day-old nondiapause larvae that had been pulse-labeled with [³H]leucine showed that the half life of lipophorin synthesis and processing was about 40 minutes. Release of total protein and lipophorin from the fat body of 13 day-old nondiapause larvae into Grace's medium was inhibited by 56 and 60%, respectively, when 10 μg/ml tunicamycin was incorporated into medium.     

Vitellogenin synthesis in female larvae of the gypsy moth,Lymantria dispar (L.): suppression by juvenile hormone

• 1.1. Fat body from feeding-phase, last instar gypsy moth females incorporates l-[³⁵S]methionine in vitro into two vitellogenins with the same molecular masses (165 and 180 kDa) as the apo-vitellogenins found in teh hemolymph and the apo-vitellins in teh eggs.
• 2.2. Both apo-vitellogenins are observed in the medium of fat body cultures, but only the 180 kDa apo-vitellogenin is observed in extracts of cultured tissue.
• 3.3. Synthesis and accumulation of the apo-vitellogenins are suppressed in a dose-dependent manner by topical treatment with the juvenile hormone analog, methoprene, prior to day 4.
• 4.4. This suppression suggests that a declining juvenile hormone titre is involved in the initiation of vitellogenin synthesis.

     

Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.     

Juvenile hormone binding components of locust fat body

Juvenile hormone (JH) binding components from the fat body of the African migratory locust were analyzed in a search for a potential nuclear JH receptor. Biosynthetically prepared 10R[³H]JH III gave a high proportion of specific binding to isolated nuclei and extracted proteins; data obtained with the JH analogs, [³H]methoprene and [³H]pyriproxyfen, on the other hand, were obscured by abundant non-specific binding. The vast majority of the high affinity JH III binding activity present in cytosolic and nuclear extracts was due to a high molecular weight JH binding protein (JHBP) which has previously been identified in locust hemolymph. This protein has several chromatographic forms which interfered in the search for a nuclear JH receptor. When specific antiserum was used to remove JHBP from nuclear extracts, a novel JH binding activity (NBP) was detected. NBP could be separated from JHBP by precipitation with ammonium sulfate. NBP displayed a high affinity for JH III (Kd = 0.25 nM) and JH I and JH II competed strongly for JH III binding, whereas methoprene and pyriproxyfen showed apparent competition when present in 1,000-fold excess. NBP was present in nuclear extracts at approximately 25,000 sites per cell; levels were similar in male and female locusts and were not greatly affected by the presence or absence of JH. The characteristics of NPB make it a strong candidate for a nuclear JH receptor. © 1995 Wiley-Liss, Inc.     

A novel protein produced by the vitellogenic fat body and accumulated in mosquito oocytes

Summary The fat body of vitellogenic mosquitoes was found to synthesize and secrete another protein in addition to vitellogenin, that accumulated in developing oocytes. In the tissues, this protein has M_r = 53000 on SDS-PAGE under reducing or non-reducing conditions. This protein is glycosylated as shown by [³H]mannose incorporation and experiments with tunicamycin. Polyclonal antibodies were produced using the ovarian 53-kDa peptide. Immunoblot analysis demonstrated the immunological identity of 53 kDa peptides from the fat body and the ovary. Furthermore, the 53-kDa protein (53KP) is synthesized and secreted exclusively by the vitellogenic fat body. Radioimmunoassay showed that 53KP is produced by the female fat body as early as 4 h and reaches its peak near 24 h after the initiation of vitellogenesis. Synthesis then drops to low levels by 36 h and declines to background levels by 48 h. In vitro experiments conducted on fat bodies of previtellogenic females demonstrated that the synthesis and secretion of 53KP can be stimulated by a physiological dose of 20-hydroxyecdysone (10^–6 M). Immunocytochemical studies of the ovary demonstrate that 53KP is present in channels between follicle cells, in the perioocytic space and in yolk granules of the developing oocytes. This suggests that 53KP is accumulated in the oocytes by a pathway similar to that of vitellogenin.     

Purification, characterisation and titre of the haemolymph juvenile hormone binding proteins from Schistocerca gregaria and Gryllus bimaculatus

Juvenile hormone binding proteins (JHBPs) were extracted from the haemolymph of adult desert locusts, Schistocerca gregaria, and Mediterranean field crickets, Gryllus bimaculatus. The JHBPs were purified by polyethyleneglycol precipitation, filtration through molecular weight cut off filters and chromatography on a HiTrap heparin column. The juvenile hormone (JH) binding activity of the extracts was measured using a hydroxyapatite assay and the purification progress was monitored by native gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The haemolymph JHBPs of both insects are hexamers composed of seemingly identical subunits. The JHBP of the locust has a native Mr of 480 kDa with subunits of 77 kDa, whereas the JHBP of the cricket has a Mr of 510 kDa with subunits of 81 kDa. The locust JHBP binds JH III with moderate affinity (KD = 19 nM). Competition for binding of JH II and JH I was about 2 and 5 times less, respectively. The cricket JHBP also has a moderate affinity for JH III (KD = 28 nM), but surprisingly, competition for binding of JH II was equal to that of JH III and JH I competed about 3 times higher. No sequence information was obtained for the locust JHBP, but the N-terminal sequence of the cricket JHBP shows ca. 56% sequence homology with a hexamerin from Calliphora vicina. Antisera raised against the purified JHBPs were used to measure age- and sex-dependent changes in haemolymph JHBP titres and to confirm that the JHBPs of both species are immunologically different.     

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaeamaderae

The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [³H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaeamaderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Synthesis and release of proteins from cultured larval fat body of the southwestern corn borer,Diatraea grandiosella

The larval fat body of the southwestern corn borer, Diatraea grandiosella, was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [³H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [³H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [³H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.     

Biosynthesis of polyunsaturated fatty acids by the australian field cricket,Teleogryllus commodus

Aspects of testicular fatty acid biochemistry from the Australian field cricket, Teleogryllus commodus, are reported. Over 10% of the phospholipid fatty acids were C20 polyunsaturated fatty acids (PUFAs), with nearly 6% arachidonic acid (20:4). The testes and ovaries accumulated a large proportion of label from radioactive arachidonic acid that was injected into the hemocoel (about 30%). Specificity in the uptake was shown by comparison to a similar study with labelled stearic acid, in which only 1.5% of the radioactivity was taken up by testes. Sixty percent of the radioactivity taken up by testes from [³H]20:4 was incorporated into phospholipids and 30% into triacylglycerols. Fat body of males and females incorporated 27% of the [³H]20:4 into phospholipids and 68% (males) or 55% (females) into triacylglcyerols. Radioactivity from [1-¹⁴C]acetate was incorporated into testicular linoleic acid and eicosatrienoic acid, but not eicosatetraenoic acid, suggesting the de novo biosynthesis of both 18:2 and a C20 PUFA by this species. Label from injected [U-¹⁴C]linoleic acid was recovered mostly as linoleic acid, with a small portion of the recovered radioactivity in eicosatrienoic acid, but not eicosatetraenoic acid. Very little label from injected linoleic acid occurred as monounsaturated or saturated fatty acids, indicating only slight, if any, β-oxidation of 18:2 to acetate and subsequent lipid synthesis.     

The Regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog

In adult female Drosophila melanogaster an increase in the synthesis and secretion of three yolk polypeptides (YPs) occurs during the first 24 hr after eclosion. During organ culture, these same polypeptides are synthesized and secreted into the medium by both fat body and ovaries. Two hormones, 20-hydroxyecdysone (20-HE) and a juvenile hormone analog (ZR-515) stimulate synthesis and secretion of YPs into the hemolymph of isolated female abdomens. The present experiments were undertaken to compare synthesis of YPs in normal females with YP synthesis in preparations deprived of anterior endocrine glands, and to find which hormone stimulates synthesis in the different organs. Separation of hemolymph proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that at eclosion incorporation of [³⁵S]methionine into YP1 and YP2 was low and was barely detectable in YP3. Over the next 24 hr the rate of label incorporation increased for all the YPs. Isolation of female abdomens at eclosion prevented this increase in label incorporation but did not entirely abolish YP synthesis. Application of either ZR-515 or 20-HE to isolated abdomens stimulated up to ninefold label incorporation into three polypeptides which comigrated with YPs from normal vitellogenic females. The response of isolated abdomens to ZR-515 or 20-HE was first detectable between 90 and 135 min after hormone application. The stimulated bands were confirmed to be YPs by a comparison of peptide digests of each of the three labeled polypeptides with those of the yolk polypeptides from intact vitellogenic females. The hypothesis that the two hormones might act on different organs was tested by treating isolated female abdomens with various concentrations of either ZR-515 or 20-HE and then culturing the stimulated organ in vitro with [³⁵S]methionine. The fat body responded to both hormones by synthesizing and secreting into the culture medium polypeptides which comigrated with the YPs found in hemolymph, whereas the ovary produced similar polypeptides only after ZR-515. These secreted polypeptides were confirmed to be YPs by repeating the experiment using organs from heterozygotes for both YP2 and YP3 electrophoretic variants. Such organs synthesized five polypeptides which comigrated with the corresponding yolk polypeptides. These findings are discussed in relation to a hypothesis for the action of the two hormones.     

Follicle cell polyploidy in Leucophaea maderae: Regulation by juvenile hormone

Microspectrophotometric analysis of Feulgen-stained nuclei of the terminal follicle cells in the cockroach Leucophaea maderae showed that during maturation the follicle cells became polyploid. In virgin females, the follicle cell nuclei were diploid. After mating, and during vitellogenesis, the ploidy of the follicle cells increased from 2 C to 32 C with a small percentage of 64 C nuclei. There was no further increase in the ploidy levels during the chorionic stage of development.Injections of juvenile hormone III into decapitated virgin females elevated the ploidy levels in the follicle cells. The DNA content of these nuclei at 96–120 h after injection of juvenile hormone III increased from 2 C to 4 C. Such polyploidization of nuclei was dose-dependent with the highest DNA content occurring in response to 25–50 μg juvenile hormone III. The juvenile hormone-induced increase in DNA content correlated with an increase in the rate of [³H]thymidine incorporation into DNA.Our data suggest that the role of juvenile hormone in follicle cell development during the vitellogenic period, whether direct or indirect, is to promote selectively a large increase in the DNA content of the cells. This may facilitate the next stage of follicle cell development, choriogenesis.     

Assembly and secretion of lipophorin by the larval fat body of the southwestern corn borer,Diatraea grandiosella: An in vitro study

The synthesis, processing, and secretion of lipophorin by the larval fat body of the southwestern corn borer, Diatraea grandiosella, was examined using in vitro techniques. Pulse-labeling of lipophorin with [³⁵S]methionine showed that apolipophorin-I and -II were each synthesized and secreted from the fat body into Grace's medium with an intracellular transit time of about 45 min. Secretion of the apolipoproteins from the fat body became insensitive to the presence of monensin, which disrupts protein processing in the Golgi complex, at 30 min, indicating that most of the pulse-labeled apolipoprotein has transited the Golgi complex by this time. Three inhibitors of protein processing, carbonylcyanide m-chlorophenyl hydrazone, monensin, and brefeldin A, inhibited secretion of lipophorin into medium. Puromycin treatment did not appear to result in the secretion into the medium of lipophorin particles containing incomplete translation products of apolipophorin-I or -II. Incubation of fat bodies with [³H]oleate resulted in the secretion of lipophorin containing [³H]glycerides, a process that was inhibited by cycloheximide, puromycin, and monensin, indicating that apolipoprotein synthesis is required for secretion of [³H]glyceride on nascent lipophorin particles. In contrast, suramin, which has been shown to block the binding of lipophorin to plasma membrane receptors, inhibited the synthesis and secretion of lipophorin, but it did not appear to inhibit the transfer of [³H]lipid from the fat body to lipophorin. Inhibitors of protein synthesis and processing, therefore, can be used to distinguish between secretion of lipophorin-associated lipids and secretion of lipids mediated by the lipid-transfer particle outside the plasma membrane of the fat body.     

Metabolic shift from lipogenesis to glycogenesis in the last instar larval fat body of the silkworm,Bombyx mori

When [¹⁴C]glucose was injected into the last instar larvae of the silkworm, Bombyx mori, the label was incorporated into various tissues at varying degrees depending on the developmental stages. Fat body exhibited high incorporation rates throughout the feeding periods. Silk glands became active in incorporation but midgut decreased toward larval maturation. The pulse labeling experiment clearly demonstrated that the metabolic shift from lipogenesis to glycogenesis occurred in fat body at the middle of the last instar; a predominant incorporation was found in lipids when [¹⁴C]glucose was injected at the early stage, while at the late stage glycogen synthesis became most active. Incorporation into fat body proteins was not a major factor throughout the instar. Extirpation of silk glands enhanced incorporation into glycogen and proteins at the late stage but did not affect lipid synthesis. Long-term chase showed that fat body lipids and proteins synthesized at the early stage were totally carried over into the pupal fat body, while much glycogen produced at the late stage was used during the larval-pupal transformation with the remainder carried over into the pupa.From these results the metabolic shift from lipogenesis to glycogenesis in fat body is discussed in relation to the storage function of the fat body for pupal metamorphosis.     

Regulation of vitellogenin synthesis by juvenile hormone analogue in Coccinella septempunctata

Vitellogenesis in the lady beetle, Coccinella septempunctata, is controlled by juvenile hormone (JH). When immature females were reared on an artificial diet, they were characterized by hypertrophy of the fat bodies and slow development of the ovaries. Vitellogenin (Vg) synthesis in the fat body remained at a very low level throughout adult development. RNA synthesis also stayed at a relatively low level. Treatment with hydroprene induced vitellogenesis in these non-fecund females. Stimulation in Vg synthesis in hormone-treated females was demonstrated both in vivo and in fat body culture. Synthesis of fat body RNA also increased after hormone treatment. Fat body RNA from hormone-treated females directed the synthesis of Vg polypeptides both in Xenopus oocytes and in a reticulocyte lysate. Thus the induction of Vg synthesis by JH involves an increase in the level of translatable Vg mRNA.     

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.