In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say)

Injection of the hormone 20-hydroxyecdysone (20-E) into partially fed (virgin) female adults of the American dog tick, Dermacentor variabilis, while they are attached and feeding on the rabbit host, initiated the expression of the vitellogenin (Vg) gene, and Vg protein secretion and uptake by the ovary. The induction of egg production by 20-E in this bioassay was dose dependent in the range of 1-50 times the concentration normally found in a replete, vitellogenic female. Ticks examined 4 d after the 50 x treatment were still attached to the host, had numerous enlarged vitellin-filled (brown) oocytes in their ovaries, but had not engorged to repletion. The ovaries reached weights similar to those found in untreated, replete (mated) females (pre-oviposition) while solvent-injected controls demonstrated no increase in oocyte size or increase in ovary weight. An increase in the levels of a putative Vg protein was observed in hemolymph samples collected 1, 2 and 3d post-20-E injection but was not observed in the corresponding solvent controls as determined by native PAGE. Analysis of the ecdysteroid-induced protein by tryptic digestion-mass fingerprinting and BLASTP found that the putative Vg had the strongest match to GP80 (U49934), the partial sequence for the vitellogenin protein from Boophilus microplus. A partial Vg cDNA was cloned and sequenced from replete females of D. variabilis with a high similarity to GP80. Using this message as a probe, Northern blots conducted with RNA collected from partially fed, virgin females 1, 2 and 3d post-20-E injection showed upregulation of the Vg mRNA on all 3 days. Controls injected with solvent only showed no Vg mRNA. Injections with juvenile hormone III did not stimulate Vg expression, oocyte growth or full engorgement. These studies indicate that ecdysteroids and not JH can initiate expression of the Vg gene, Vg protein synthesis and release into hemolymph, and Vg uptake into developing oocytes under bioassay conditions mimicking normal feeding on the host.     

Physical characterization and sequence identification of the ovary maturating parsin. A new neurohormone purified from the nervous corpora cardiaca of the African locust (Locusta migratoria migratorioides.

A novel neurohormone, which anticipates ovarian maturation, was recently purified using liquid chromatography from the African locust nervous corpora cardiaca. Both its function and production by the pars intercerebralis of Locusta migratoria lead to its name, the ovary maturating parsin (Lom OMP). In this study, the Lom OMP was physically and chemically characterized. Its multiply charged ion spectrum was interpreted as two peaks of quite equal size having molecular masses of 6923.4 Da (major peak) and 6907.3 Da. The Lom OMP presented no periodic secondary structure according to the far ultraviolet circular dichroism spectrum obtained. It is composed of 65 amino acids and included a high concentration of alanine but is devoid of cysteine, isoleucine, methionine, lysine and threonine. The amino acid sequence indicated only one microheterogeneity, observed at position 26, consisted in the replacement of serine by alanine. The calculated Mr of the two acidic isoforms (calculated pHi = 4.87) were found to be in agreement with mass spectrometry measurements. When compared to the sequence libraries, the Lom OMP, the first insect gonadotropic neurohormone, was revealed as an unique protein.     

Vitellogenin mRNA in locust fat body: coordinate induction of two genes by a juvenile hormone analog

Levels of vitellogenin (Vg) mRNA in Locusta migratoria fat body were determined as indicators of gene expression induced by the juvenile hormone analog methoprene. After injection of methoprene into juvenile hormone-deprived locusts, excised fat bodies were cultured with [3H]leucine for immunochemical assay of Vg synthesis, and RNA was assayed for Vg mRNA content by hybridization with probes from the previously cloned locust Vg genes A and B. In general, the rise in Vg mRNA paralleled the rise in Vg synthesis. During the primary response to methoprene (in female locusts in which the corpora allata had been destroyed immediately after emergence), Vg mRNA was first detected after 18-24 hr and accumulated rapidly between 36 and 48 hr. The secondary response (in locusts allatectomized during vitellogenesis and kept until Vg disappeared) was accelerated, as Vg mRNA was detectable at 12 hr and titers rose steeply after 18 hr. When Vg synthesis was prematurely induced by injection of methoprene into fifth-stage female larvae, the kinetics of mRNA accumulation were similar to those of primary stimulation in the adult. After allatectomy of vitellogenic females, fat body Vg mRNA decayed with a half-life of about 24 hr, roughly paralleling the decline in Vg synthesis. Assays with the two Vg probes showed coordinate accumulation of gene A and gene B messages under all conditions tested: during primary and secondary stimulation in adult females and in the low-level response obtained by treating male larvae with methoprene.     

Juvenile hormone regulation of vitellogenin synthesis in the red flour beetle,Tribolium castaneum

To elucidate the endocrine regulation of vitellogenin (Vg) synthesis in the red flour beetle, Tribolium castaneum, the titers of juvenile hormone (JH) and ecdysteroids in the whole body of female beetles were measured and compared with Vg mRNA levels. Juvenile hormone levels remained high while the ecdysteroid levels declined steadily during 1–5 days post adult emergence (PAE). The Vg mRNA levels began to increase by the end of 3rd day PAE and peaked by the 4th–5th day PAE. Gene expression profiling by microarray and quantitative real-time PCR analyses of RNA isolated from 1 to 5 days PAE beetles revealed that the genes coding for proteins involved in JH biosynthesis and action, but not those involved in 20-hydroxyecdysone (20E) biosynthesis and action had similar expression patterns as the genes coding for Vg. RNA interference (RNAi)-aided knock-down in the expression of these genes showed that both JH and 20E were required for Vg gene expression. However, Vg mRNA was induced by the application of JH III but not by the injection of 20E into the previtellogenic females. These data suggest that JH is required for Vg synthesis in the fat body and 20E influences Vg synthesis through its action on oocyte maturation.     

Edysteroid receptor (EcR) shows marked differences in temporal patterns between tissues during larval-adult development in Rhodnius prolixus: correlations with haemolymph ecdysteroid titres

The presence of ecdysteroid receptor (EcR) in various tissues was studied throughout larval-adult development of the blood-sucking bug, Rhodnius prolixus, using an antibody to EcR that recognizes all isoforms. On Western blots, the antibody recognizes three peptides of approximate molecular masses of 70, 68 and 64 kDa, from epidermis and fat body of developing larvae, which contain high levels of haemolymph ecdysteroids. These peptides are absent from both unfed larvae and adults, which are devoid of ecdysteroids. In vitro treatment of epidermis and fat body from unfed larvae with 20E induces the appearance of all three EcR immunoreactive peptides. The stage-specific appearance and 20E inducibility of the peptides implies that they represent the native EcR(s) of Rhodnius. Confocal fluorescence analysis using this antibody revealed a great diversity of temporal profiles of EcR in various tissues during development. Developmental profiles of EcR were examined in abdominal epidermis, fat body, spermatocytes, brain (including the medial neurosecretory cells), prothoracic glands (PGs), rectal epithelium and Malpighian tubules. EcR fluorescence was confined to the nuclei in close association with chromatin. EcR was absent from tissues of unfed larvae or adults, supporting the results from Western blots. Different tissues develop EcR at different developmental times and in the presence of radically different concentrations of haemolymph ecdysteroids, retain EcR for different lengths of time and lose EcR at different concentrations of ecdysteroids. These results suggest that each tissue possesses a distinctive response mechanism to ecdysteroids. An exception to this, are the PGs, which exhibited no EcR fluorescence at any time during development.     

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.     

Entomogenous fungus Nomuraea rileyi inhibits host insect molting by C22-oxidizing inactivation of hemolymph ecdysteroids

The entomogenous fungus Nomuraea rileyi reportedly secretes a proteinaceous substance inhibiting larval molt and metamorphosis in the silkworm Bombyx mori. We studied the possibility that N. rileyi controls B. mori development by inactivating hemolymph molting hormone, ecdysteroids. Incubation of ecdysone (E) and 20-hydroxyecdysone (20E) in fungal-conditioned medium resulted in their rapid modification into products with longer retention times in reverse-phase HPLC. Each modified product from E and 20E was purified by HPLC, and identified by NMR as 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone. Some other ecdysteroids with a hydroxyl group at position C22 were also modified. Injection of the fungal-conditioned medium into Bombyx mori larvae in the mid-4th instar inhibited larval molt but induced precocious pupal metamorphosis, and its injection into 5th instar larvae just after gut purge blocked pupal metamorphosis. In hemolymph of injected larvae, E and 20E disappeared and, in turn, 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone accumulated. These results indicate that N. rileyi secretes a specific enzyme that oxidizes the hydroxyl group at position C22 of hemolymph ecdysteroids and prevents molting in B. mori larvae.     

Effect of allatectomy on ecdysteroid-dependent development of Rhodnius prolixus larvae

the regulation of haemolymph titres of ecdysteroids during larval development of the bloodsucking bug, Rhodnius prolixus was studied. Corpus allatum ablation in 4th-instar larvae 1 day after feeding was reflected in an increase of the intermoult period and in a high level of ecdysial arrest. These effects could be corrected by juvenile hormone and ecdysone therapies. Comparison of the ecdysteroid titres in haemolymph determined in control and allatectomized larvae, at different intervals after feeding, showed that allatectomy drastically depressed the ecdysteroid levels. Juvenile hormone treatment reestablished ecdysteroid titres in the haemolymph of allatectomized insects. Isolated prothoracic glands from allatectomized larvae had a very low production of ecdysteroid-RIA-activity when compared with prothoracic glands from control or allatectomized larvae which received in vivo juvenile hormone treatment. The complexity of the corpus allatum-prothoracic glands interaction in Rhodnius post-embryonic development is discussed.     

Ecdysteroids during oögenesis in the ovoviviparous cockroach Nauphoeta cinerea

By using thin-layer chromatography and high-pressure liquid chromatography combined with radioimmunoassay as well as gas chromatography-mass spectrometry we have identified and quantified ecdysteroids in ovaries and haemolymph of adult female Nauphoeta cinerea. Our analyses demonstrate the presence of ecdysone and 20-hydroxyecdysone, the latter being clearly predominant in all stages investigated. Titre determinations of free ecdysteroids in ovaries show that the 20-hydroxyecdysone concentration is highest (approximately 400 ng/g) at the beginning of chorion formation, suggesting an involvement in this process. Towards ovulation, the titre of free ecdysteroid drops and is low in the newly ovulated egg case. Measurement of immunoreactive highly polar products demonstrates that their concentration remains on a low level throughout the oöcyte maturation period; hydrolysis experiments with Helix pomatia enzymes reveal that, compared to the free ecdysteroids in the ovary, only small quantities of ecdysteroids are present as Helix hydrolysable conjugates. If one compares the quantities of free ecdysteroids in the ovary with those in the haemolymph it becomes apparent that the concentration in the haemolymph is about 10 times lower than that in the ovary.In vitro incubation of follicle cells from oöcytes at stages around chorion formation reveals that these cells are able to produce ecdysone and 20-hydroxyecdysone, and incubation with [³H]-ecdysone demonstrates that ecdysone is efficiently converted to 20-hydroxyecdysone in a stage-dependent manner. These observations strongly suggest that the follicle cells are the site of ecdysteroid biosynthesis and of C-20-ecdysone hydroxylation.A comparison of these findings with observations made of other insects such as locusts and mosquitoes demonstrates significant differences in quality, composition, titre fluctuation and distribution of ecdysteroids in adult females from different species and suggests that these ecdysteroids might fulfil multiple and various biological functions.     

Regulation of vitellogenesis in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)

Studies were undertaken to investigate vitellogenesis and its regulation in female adults of the fall armyworm, Spodoptera frugiperda. A single female-specific protein, likely to be the S. frugiperda vitellogenin (Vg), appeared approximately 5 h after adult eclosion in the hemolymph of virgin females. The concentration of the protein increased with age as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed. A protein with the same relative molecular mass was also present in egg extracts, but absent from hemolymph samples from male moths. The relative molecular mass of the designated S. frugiperda Vg was determined as 164.5+/-2.5 kDa. Vitellogenic oocytes became visible 36-48 h after emergence and egg deposition began on day 3 of adult life. Vg could not be detected in the hemolymph of females decapitated directly after eclosion. When decapitated virgin females were injected with the JH-mimic methoprene (MP), the level of Vg was comparable to that in non-decapitated moths, indicating that vitellogenesis in S. frugiperda depends on juvenile hormone (JH). However, the number of vitellogenic oocytes was somewhat lower than in non-decapitated virgin females. Injection of 20-hydroxyecdysone (20E) promoted Vg production to a similar extent in decapitated female moths, but in contrast to methoprene injection, treatment with 20E never resulted in the production of vitellogenic oocytes. In vitro cultivated ovaries of adult females dissected directly after eclosion produced lower amounts of ecdysteroids than those isolated on day 1 after emergence. Our results suggest a crucial role for 20E in the induction of vitellogenesis in the noctuid S. frugiperda, while JH seems to be essential for the continued uptake of Vg by developing oocytes and may trigger 20E biosynthesis in the ovary.     

Stimulation of vitellogenin production by methoprene in prepupae and pupae of manduca sexta

Denaturing electrophoresis of hemolymph from prepupae of M. sexta showed trace amounts of polypeptides with mobilities corresponding to those of vitellogenin (Vg) apoproteins from adult females. Absence of the polypeptides in allatectomized insects suggested regulation by juvenile hormone (JH). Daily administration of 10 μg of the JH analog methoprene from day 4 of the fifth stage to day 0 of the pupal stage caused accumulation of these polypeptides. They were identified as apovitellogenins (apoVgs) immunochemically with Vg antiserum. Stimulation of Vg in response to methoprene varied with age. In all cases, day 0 female pupae were highly responsive. Vg synthesis was not stimulated when pupae were injected with 20-hydroxyecdysone (20-HE) in addition to methoprene. Methoprene-stimulated Vg synthesis was also abolished by inhibitors of mRNA or protein synthesis (α-amanitin, actinomycin, cycloheximide). This result indicated that methoprene-stimulated Vg accumulation requires gene expression. A Vg cDNA (2.1 kb) obtained by immunoscreening of the λgt 11 library, when used as a radiolabelled probe, hybridized with a 5.1 kb mRNA from total RNA of female fat body. It also hybridized with fat body RNA of normal prepupae and methoprene treated day 0 pupae but not with that of early fifth instars or solvent control pupae. The results indicate that the trace amounts of Vg found in prepupal stages are due to a weak expression of the Vg gene, which is stimulated by JH and repressed by 20-HE. © 1994 Wiley-Liss, Inc.     

Juvenile hormone-controlled vitellogenin synthesis in the fat body of the locust (Locusta migratoria): Isolation and characterization of vitellogenin polysomes and their induction in vivo

With the aid of EGTA to control the endogenous RNase activity, a predominant population of heavy polysomes was isolated from the fat body of reproductively active female locusts. The identity of these as vitellogenin (Vg)-synthesizing polysomes was established by precipitation of the associated nascent polypeptides with anti-vitellin serum and by translation of the polysomal RNA in Xenopus laevis oöcytes. By EM observation these polysomes were found to contain 40–50 ribosome monomers.In normal development, accumulation of ribosomes in the fat body of female locusts began at about day 6 after adult ecdysis (terminal oöcyte = 2 mm or smaller), and was followed by the appearance of Vg-polysomes beginning at 3 mm terminal oöcyte length (about day 8–10 after adult ecdysis). The content of Vg-polysomes reached a maximum at 5–6 mm oöcyte length (about day 14), and then fell to an undetectable level as the oöcytes reached their maximum size (7 mm).In allatectomized females, Vg-polysomes were induced by treatment with an active juvenile hormone analogue, ZR-515. A single application with ZR-515 produced a massive accumulation of ribosomes and light polysomes in the first 48 hr which was followed by the rapid formation of Vg-polysomes to give a maximum at 72 hr. After the decay of this effect a second dose of ZR-515 resulted in the rapid appearance of Vg-polysomes without the initial generation of ribosomes.     

Ovarian follicle cells are the site of vitellogenin synthesis in the Pacific abalone Haliotis discus hannai

Only a few biochemical and molecular studies on yolk proteins (vitellins) have been carried out in mollusks, mainly in bivalves, while information on prosobranch vitellogenesis is still limited. In this study, we cloned a full-length cDNA encoding vitellogenin (Vg) in the Pacific abalone Haliotis discus hannai. The complete Vg cDNA consists of 7753 nucleotides with a long open reading frame encoding 2391 amino acid residues. The deduced primary structure contains the N-terminal amino acid sequences of the 95 kDa and 150 kDa subunits of vitellin of the abalone and shows similarities to Vgs of other mollusk, fish, nematode and coral species. In common with bivalve Vgs, the abalone Vg gene was expressed only in the ovary. In situ hybridization analysis further localized Vg mRNA to the follicle cells in the ovary. We conclude that the follicle cells are the site of Vg synthesis in H. discus hannai.     

Juvenile hormone and ovarian maturation in the diptera: A review of recent results

The roles of the JHs and ecdysteroids in ovarian maturation of adult Aedes aegypti, A. atropalpus, Musca domestica and Drosophila melanogaster have been characterized by comparing the effects of surgical (allatectomy, ovariectomy, decapitation, abdominal ligation), genetic, and nutritional (sugar feeding) manipulations. The results show that in all species JHs, or their mimics, and 20-hydroxyecdysone act in combination to stimulate ovarian maturation and vitellogenin (Vg) synthesis and that high doses of exogenous JHs, or their mimics, stimulate ovarian ecdysteroid synthesis, at least in A. aegypti, A. atropalpus, and D. melanogaster. Thus the gonadotropic regulatory mechanisms that exist in various dipteran species are more similar than originally suspected. In M. domestica, and possibly D. melanogaster, 20-hydroxyecdysone is present in the hemolymph at vitellogenic levels in newly emerged females and may persist in ovariectomized adults. If true for D. melanogaster, this could explain why topical application of JH, or its mimics, to ovariectomized, isolated abdomens is effective in stimulating Vg synthesis in the absence of the ovaries.     

Dynamics of vitellogenin mRNA expression during vitellogenesis in the banana shrimp Penaeus (Fenneropenaeus) merguiensis using real-time PCR

An open reading frame (ORF) of vitellogenin (Vg) cDNA was amplified from the ovaries of the banana shrimp, Penaeus merguiensis. An examination of Vg-deduced amino acid sequence revealed the presence of cleavage sites at a consensus motif for subtilisin-like endoproteases prior to the N-terminal sequences of purified vitellin (Vt) subunits. A comparison of the primary structures of Vg molecules in decapod crustacean species revealed the existence of a common characteristic structure, and phylogenetic analysis reflected the current taxonomic classifications of crustaceans. A PCR product of 1.1 kb encoding the 3'-end of Vg cDNA was cloned from the hepatopancreas. Although its sequence was almost identical to that of the same region of the ovarian Vg, with only 18 nucleotide differences, analysis suggests that they have been subjected to natural selection, indicating that there may be two different, tissue-specific Vg genes in P. merguiensis. This is consistent with the different expression patterns of Vg mRNA, as determined by real-time PCR. Vg mRNA levels were maintained at low levels during the previtellogenic stage and they increased as vitellogenesis progressed to reach a peak at the early vitellogenic stage in the ovary or at the vitellogenic stage in the hepatopancreas, and thereafter, levels decreased. Expression of Vg mRNA was much higher in the ovary compared to the hepatopancreas at all stages of ovarian development, implying that the ovary is mainly responsible for Vt synthesis. These indicate that penaeids constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.     

Vitellogenin expression in queen ovaries and in larvae of both sexes of Apis mellifera

In the honeybee, Apis mellifera, vitellogenin (Vg) expression has been detected in the ovary of queens, but not in that of workers. In addition, larvae of both sexes produce Vg in significant amounts, which suggest that Vg serves for functions additional to oocyte growth and energy supply to the embryo. In vivo hormone treatment experiments suggest that the decrease of 20-hydroxyecdysone concentration occurring in previtellogenic phases allows Vg production. Southern analysis indicates that the Vg gene is present as a single copy in the honeybee genome.     

Programmed cell death of larval tissues induced by juvenile hormone in the bamboo borer, Omphisa fuscidentalis

Programmed cell death (PCD) plays a critical role during animal development through the destruction of unneeded cells and tissues. In some insects, the prothoracic glands (PGs) and anterior silk glands (ASGs) are larval-specific tissues that are normally eliminated by PCD after pupation. Previous studies report that juvenile hormone analog (JHA) terminates the larval diapause of Omphisa fuscidentalis by increasing the hemolymph ecdysteroids that trigger PCD. Because JHA may indirectly induce the PCD of the PGs and ASGs of Omphisa diapausing larvae, the effects of JHA on the induction of PCD were determined. The application of 1μg JHA induced PCD in the PGs and ASGs of larvae identified as stage G0 (prior to pupation). The injection of 1μg 20E triggered the PCD of the ASGs when the larvae expressed a G0-G1 morphology, whereas PCD occurred in the PGs on day 1 post-injection. Histological studies revealed similar patterns of morphological changes during the PG and ASG PCD in the JHA- and 20E-treated larvae. Furthermore, to confirm that PCD was induced by a high ecdysteroid level that increases after JHA application, the expression profiles of EcR-A and EcR-B1 in the PGs and ASGs from the JHA-treated larvae were examined, and the results showed that the expression levels of EcR-A and EcR-B1 mRNA increased during the G0 stage. These results suggest that JHA may be involved in PCD by increasing the ecdysteroid titer, leading to termination of the larval diapause period in Omphisa fuscidentalis.     

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.     

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.     

Effect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum

Earlier work from our laboratory indicated that injection of 20-hydroxyecdysone (20E) into non-vitellogenic female Amblyomma hebraeum ticks stimulates the synthesis of vitellogenin (Vg), but not its uptake into oocytes [Friesen, K., Kaufman, W.R., 2004. Effects of 20-hydroxyecdysone and other hormones on egg development, and identification of a vitellin-binding protein in the ovary of the tick, Amblyomma hebraeum. Journal of Insect Physiology 50, 519-529]. In contrast, Thompson et al. [Thompson, D.M., Khalil, S.M.S., Jeffers, L.A., Ananthapadmanaban, U., Sonenshine, D.E., Mitchell, R.D., Osgood, C.J., Apperson, C.S., Roe, M.R., 2005. In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say). Journal of Insect Physiology 51, 1105-1116] demonstrated that injection of 20E into virgin female Dermacentor variabilis ticks stimulated both vitellogenesis and Vg uptake into oocytes. In addition to the species difference in the two studies there were substantially different methods for injecting 20E. In our earlier work we injected small partially fed ticks after removing them from the host. Thompson et al. injected the females while they remained attached to the host. So in this study we repeated our earlier experiments on A. hebraeum using on-host injection. We also injected 20E into off-host ticks with or without haemolymph collected from engorged ticks (days 2-10 post-engorgement), or from large partially fed mated ticks in the rapid phase of engorgement, to see whether we might detect a 'vitellogenin uptake factor' (VUF) in haemolymph. Off-host injection of 20E (0.45mug/g body weight (bw)) did not induce ovary development beyond that of vehicle-injected controls. But ticks in this study, receiving 20E plus haemolymph from engorged ticks, showed a significant increase in ovary weight beyond that of 20E alone (1.31+/-0.05% bw; 34 for 20E plus haemolymph and 1.03+/-0.05% bw; 25 for 20E alone). However, in normal engorged A. hebraeum, the ovary exceeds 7% bw at the onset of oviposition. As in our earlier work, in this study 20E stimulated Vg-synthesis (3.9+/-0.5mgVt-equivalents/ml) beyond that occurring in vehicle-injected ticks (0.76+/-0.14mgVt-equivalents/ml), and there was a further increase in ticks injected with 20E plus haemolymph from engorged ticks (8.9+/-1.0mgVt-equivalents/ml). On-host injection of 20E alone (6mug20E/g bw) did not produce a statistically significant increase in oocyte length over that of vehicle-injected controls, whereas on-host injection of 20E plus engorged haemolymph resulted in significantly larger oocytes (261+/-57mum) compared to vehicle-injected controls (132+/-11mum), compared to 20E alone (131+/-12mum), or haemolymph alone (124+/-24mum). There was a marked stimulation of Vg-synthesis by 31mug20E/g bw (6.0+/-1.5mgVt-equivalents/ml) compared to vehicle-injected controls (1.02+/-33mgVt-equivalents/ml). Vt accumulation by ovaries was significantly greater in ticks treated with haemolymph (12+/-3mugVt/mg ovary) or 20E plus haemolymph (56+/-26mugVt/mg ovary) compared to vehicle-injected controls (5.1+/-1.5mugVt/mg ovary). There was also a significant effect of 6mug20E/g bw plus engorged haemolymph on ovary weight (1.74+/-0.29% bw) compared to vehicle-injected ticks (0.95+/-0.10% bw), but not compared to ticks injected with 20E alone (1.25+/-0.19% bw). We conclude that at least some of the differences observed between the two laboratories relate to the species difference, and that there is some evidence that the engorged haemolymph of A. hebraeum contains a VUF.