The early gene Broad is involved in the ecdysteroid hierarchy governing vitellogenesis of the mosquito Aedes aegypti

The broad (br) gene, encoding a family of C2H2 type zinc-finger DNA-binding proteins, has been shown to act as a crucial member of the 20-hydroxyecdysone (20E) regulatory hierarchy in the fruitfly, Drosophila melanogaster and the moth, Manduca sexta. In this study, we have shown that the br gene is involved in the 20E-regulatory hierarchy controlling vitellogenesis in the mosquito, Aedes aegypti. Unlike E74 and E75 early genes, expression of br was activated in previtellogenic females, during a juvenile hormone (JH)-dependent period. The levels of Z1, Z2 and Z4 isoform mRNA were elevated in the fat body of 2-day-old females after in vitro exposure to JH III. However, JH III repressed 20E activation of br in 3-to 5-day-old females, indicating a switch in hormonal commitment. Expression of Z1, Z2 and Z4 was stimulated after blood feeding in both vitellogenic tissues, the fat body and the ovary, corresponding to peaks of ecdysteroid titers. In the fat body, the mRNA profiles of these three isoforms correlated well with those of yolk protein precursor (YPP) genes. These BR isoforms were activated by 20E in fat bodies cultured in vitro and behaved as early genes, with a self-repressive autoregulatory loop that can be blocked by the protein inhibitor, cyclohexamide. Multiple binding sites for all four BR isoforms were present in the 5'-regulatory region of the major YPP gene, vitellogenin (Vg). Effects of BR isoforms on the expression of Vg have been demonstrated by cell transfection analysis. In particular, BR isoforms by themselves had no effects on the Vg promoter. However, Z1 and Z4 each repressed Aedes aegypti ecdysone receptor (EcR)/Ultraspiracle (USP)-mediated 20E activation of the Vg promoter, while Z2 enhanced activation of the Vg promoter by AaEcR/AaUSP in the presence of 20E. Z3 had no obvious effect in the same experiment. These results suggested that BR isoforms are essential for proper activation and termination of the Vg gene in response to 20E. Overall, our study implicated br in the regulation of mosquito vitellogenesis.     

Distinct roles of isoforms of the heme-liganded nuclear receptor E75, an insect ortholog of the vertebrate Rev-erb, in mosquito reproduction

Mosquitoes are adapted to using vertebrate blood as a nutrient source to promote egg development and as a consequence serve as disease vectors. Blood-meal activated reproductive events in female mosquitoes are hormonally and nutritionally controlled with an insect steroid hormone 20-hydroxyecdysone (20E) playing a central role. The nuclear receptor E75 is an essential factor in the 20E genetic hierarchy, however functions of its three isoforms - E75A, E75B, and E75C - in mosquito reproduction are unclear. By means of specific RNA interference depletion of E75 isoforms, we identified their distinct roles in regulating the level and timing of expression of key genes involved in vitellogenesis in the fat body (an insect analog of vertebrate liver and adipose tissue) of the mosquito Aedes aegypti. Heme is required in a high level of expression of 20E-controlled genes in the fat body, and this heme action depends on E75. Thus, in mosquitoes, heme is an important signaling molecule, serving as a sensor of the availability of a protein meal for egg development. Disruption of this signaling pathway could be explored in the design of mosquito control approaches.     

Short-loop negative and positive feedback on ecdysone secretion by prothoracic gland in the tobacco hornworm, Manduca sexta

Ecdysteroid production by the prothoracic glands of the tobacco hornworm, Manduca sexta was found to be under feedback control by the ecdysteroids in hemolymph using both culture in vivo in diapausing pupae lacking the brain-corpora cardiaca-corpora allata complex and the prothoracic glands and culture in vitro. Prothoracic glands having relatively high activity in larvae, prepupae, or developing pupae were inhibited by ecdysone or 20-hydroxyecdysone. By contrast, prothoracic glands with low activity from feeding larvae, day 1 non-diapausing pupae and diapausing pupae were activated by both ecdysone and 20-hydroxyecdysone in vivo and in vitro. Dose-response studies on diapausing pupal glands showed that ecdysone was the most effective activator. These findings suggest that prothoracic glands are either stimulated or inhibited by ecdysone or 20-hydroxyecdysone, depending on both the secretory activity of the gland and the effective level of ecdysteroids in hemolymph. Thus, when the glands are first activated, the ecdysteroids that are secreted show a positive feedback on the glands to increase ecdysteroid output. Then the activated glands are turned off by the increasing 20-hydroxyecdysone titer in the hemolymph leading to the rapid decrease in ecdysteroid titer at the end of the molt period.     

Ecdysteroid fluctuations during embryogenesis in the giant freshwater prawn, Macrobrachium rosenbergii

Ecdysteroid levels during the embryogenesis of the giant freshwater prawn, Macrobrachium rosenbergii, were determined by radioimmunoassay and high-performance liquid chromatography. Ecdysteroids consisting of significant amounts of 20-hydroxyecdysone and high-polarity products (HPP) and lesser amounts of ecdysone and low-polarity products (LPP) were detected in mature ovaries and newly laid eggs. All ecdysteroid groups decreased gradually during the nauplius phase. With the formation of the compound eye and the appearance of the carapace and other body-like structures, marking morphogenesis to the zoeal stage, embryos showed the beginning of a continuous and dramatic increase in ecdysteroid concentrations sustained until larval hatchout. Ecdysteroid levels at hatchout were above 20-fold greater than ecdysteroid levels in newly laid eggs. More specifically, HPP and 20-hydroxyecdysone increased concomitantly, with a decrease in 20-hydroxyecdysone only at the end of the embryogenic period, while ecdysone and LPP levels remained low or undetectable. It may be postulated that the presence of ecdysteroids in ovaries and eggs represents a reserve of maternal ecdysteroids which are necessary at the commencement of embryonic development; with the differentiation of embryonic tissue capable of ecdysteroid synthesis, ecdysteroids increase rapidly to play a role in later embryonic development.     

Ecdysteroid titer during larval-pupal-adult development of the tobacco hornworm, Manduca sexta

The ecdysteroid titer during the fourth (penultimate) and fifth larval instars of Manduca sexta was characterized by large increases lasting approximately 24 and 60 hr, respectively, and these peaks occurred just before ecdysis. During the last larval instar, an earlier, smaller increase in the titer was also observed lasting approsimately 20 hr and is the increase presumed responsible for commitment to pupal development. After pupation, the ecdysteroid titer increases again, reaching a maximum between Days 7 and 9. At Day 10 postpupation, the titer declined dramatically and stabilized at ～day 14. During pupal-adult development, there was no sexual dimorphism in the ecdysteroid titer. However, a difference in the ecdysteroid titer between males and females was observed after adult eclosion. Qualitative analysis of the ecdysteroid RIA activity revealed the presence of only ecdysone and 20-hydroxyecdysone at varying ratios depending on developmental stage. For the large peaks preceding larval and pupal ecdysis, 20-hydroxyecdysone was the predominant hormone, while ecdysone was the major component during pupal-adult development. The fifth instar commitment peak was comprised of approximately equal quantities of the two ecdysteroids.     

Epidermis as the source of ecdysone in an argasid tick.

Various tissues excised from nymphs of the tick Ornithodoros parkeri at the time of epicuticle deposition were incubated in vitro. The medium from the incubation of salivary glands, coxal glands, synganglion, testis, midgut, and fat body associated with tracheal trunk showed little or no ecdysteroid immunoreactivity. Only medium from incubated integument contained ecdysteroids. The following evidence indicated that epidermal cells are the source of ecdysone: (i) when dorsal and/or ventral integuments were incubated separately, both produced ecdysteroid immunoreactive material during the course of incubation. As compared with the ecdysteroid content in the integument before incubation, the amount of ecdysteroids produced after a 24-h incubation increased 4- to 7-fold; (ii) enzymatic hydrolysis showed that neither highly polar ecdysteroid conjugates nor apolar conjugates were stored in the integument; (iii) histological and scanning electron microscope observations demonstrated that these excised integuments consisted of newly deposited epicuticle and epidermis as well as some fat body cells; (iv) HPLC RIA showed that the integument with associated fat body produced ecdysone and 20-hydroxyecdysone, while the integument produced only ecdysone after removing fat body. Presumably, ecdysone secreted by epidermis was converted into 20-hydroxyecdysone by fat body.     

Development of microsomal cytochrome P-450 monooxygenases during the last larval instar of the locust,Locusta migratoria: Correlation with the hemolymph 20-hydroxyecdysone titer

Several enzyme activities were measured in microsomes from Malpighian tubules and from fat body of the locust, Locusta migratoria, during the last larval instar and the 20-hydroxyecdysone titer was determined in the hemolymph. Ecdysone 20-monooxygenase, the cytochrome P-450 dependent monooxygenase which converts ecdysone to the active molting hormone 20-hydroxyecdysone had a low activity in the beginning of the instar, but showed a peak in both Malpighian tubules and fat body which coincided with the peak of 20-hydroxyecdysone in the hemolymph. The varying ratios of ecdysone and 20-hydroxyecdysone in L. migratoria hemolymph may therefore be accounted for by these changes in ecdysone 20-monooxygenase activity. The amounts of cytochrome P-450 and the activity of NADPH-cytochrome c reductase also showed a peak on day 5 of the instar, as did the activity of cytochrome P-450 linked lauric acid ω-hydroxylase in fat body microsomes. In larvae experimentally deprived of molting hormone, the activities of the cytochrome P-450 monooxygenases were low. The possible role of ecdysteroids in the control of developmental changes of cytochrome P-450 monooxygenases is discussed.     

Ecdysteroids in the embryos and sera of the crabs, Cancer magister and C. anthonyi

Ecdysteroids in the embryos and sera of ovigerous brachyuran crabs, Cancer magister and C. anthonyi, were measured and characterized by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC). C. magister embryos displayed a biphasic pattern of ecdysteroid fluctuation during development; titers decreased until mid embryogenesis and then increased and peaked prior to hatching. HPLC-RIA analysis indicated increasing ecdysone concentrations from mid embryogenesis to hatching. Endogenous biosynthesis of ecdysone by the embryos is suggested. In contrast, ecdysteroid titers in the embryos of C. anthonyi showed a steady decrease from very high initial concentrations. The decrease in titers of ecdysone and 20-hydroxyecdysone is suggestive of utilization of maternally derived ecdysteroids rather than endogenous biosynthesis during the shorter embryogenic period for C. anthonyi. Ecdysteroid concentrations did not differ with respect to location of the embryo within the egg mass. Serum ecdysteroids in C. magister females generally showed a monotonic pattern during brooding. However, for C. anthonyi females, increasing and decreasing titers were observed during the brood and interbrood periods, respectively. These fluctuations suggest mobilization of the ecdysteroids to the ovaries for subsequent storage and utilization during embryogenesis. The evolutionary significance of these differing patterns of ecdysteroid metabolism in these congeners is discussed.     

Metabolism of ecdysteroids during the vitellogenesis of the tick Ornithodoros moubata (Ixodoidea, Argasidae): accumulation of apolar metabolites in the eggs

The fate of injected [3H]ecdysone [( 3H]E) and 20-hydroxy-[3H]ecdysone [( 3H]20E) has been investigated in the female tick Ornithodoros moubata (Murray, 1877; sensu Walton, 1962). When injected into fed mated vitellogenic females, [3H]E is converted into [3H]20E and two apolar classes of metabolites, AP1 and AP2. Injected [3H]20E is directly converted into AP1 and AP2. AP2 is incorporated into the ovaries in a high proportion and at the end of the vitellogenic cycle represents about 25% of the total label recovered from the animal. The fate of labeled hormones injected into virgin females which perform an abortive vitellogenic cycle is quite similar. However, the ovaries incorporated less of the AP2 products. Ovaries of mated females cultured in vitro in the presence of [3H]E are able to produce [3H]20E and AP2. AP2 is incorporated, while [3H]20E is mainly found in the medium. Ovaries of virgin females presented a slower rate of transformation and of incorporation of the label. Labeled AP2 is recovered in freshly laid eggs and AP1 in the females after oviposition. AP1 and AP2 can produce [3H]20E, [3H]E, and other minor polar peaks when submitted to hydrolysis by esterase. It is concluded that the female O. moubata possesses a special enzymatic mechanism for transformation of ecdysteroids into apolar products and for selective incorporation of AP2 into the ovaries. These products are present in the freshly laid eggs and could play a role during embryogenesis.     

Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus. I. Hemolymph titers and metabolites

Hemolymph ecdysteroid (Ecd) titers were measured using radioimmunoassay (RIA) during the molt cycle of the American lobster, Homarus americanus. Individual animals showed small, transitory rises of Ecds which increased in magnitude with the onset of premolt and culminated in a large premolt peak at morphological stages D2(2)-D3(1). Male lobsters had significant postmolt peaks and late premolt titers that remained high until ecdysis. In females, postmolt peaks were absent and late premolt titers reached basal levels before ecdysis. At least seven different Ecd metabolites were identified by high-performance liquid chromatography-RIA analyses. High polarity products (HP) were the most abundant metabolites in virtually every molt stage. Titers of HP were significantly higher in males during late postmolt-early intermolt and in late premolt. Levels of 20-hydroxyecdysone (20E) were equivalent in both sexes and correlated with the morphological changes associated with premolt. Evidence was also obtained for the presence of ecdysone, ponasterone A, and other as yet unidentified metabolites. The pattern of Ecd metabolites in the hemolymph supports other data indicative of 20E as the major molting hormone. Metabolism of 20E is primarily toward more polar compounds, including conjugates.     

Ovarian development and correlated changes in hemolymphatic ecdysteroid levels in two spiders, Coelotes terrestris and Tegenaria domestica (Araneae, Agelenidae).

Oocyte development during the first vitellogenic cycle of Coelotes terrestris and Tegenaria domestica is described. Under the present conditions, this development took about 40 days during which the oocytes went through six stages of maturation. For the first time presence of ecdysteroids is reported in adult females of spiders. Hemolymphatic ecdysteroid peaks were observed in both species at the transition between previtellogenesis and vitellogenesis. 20-Hydroxyecdysone and ecdysone were present in slightly different ratios in C. terrestris as well as T. domestica. These data largely agree with current views of ovarian development in Arthropods.