Evaluation of ecdysteroid antisera for a competitive enzyme immunoassay and extraction procedures for the measurement of mosquito ecdysteroids.

Ecdysteroid hormones regulate several aspects of insect development and reproduction. The predominant ecdysteroids produced by insects including mosquitoes are ecdysone (E) and 20-hydroxyecdysone (20E). The ability to measure E and 20E titers is essential for many studies, but few sensitive, low cost options are currently available for doing so. To address this deficiency, we developed a new enzyme-linked immunoassay (EIA). In the first part of the study, we compared the affinity of two new antisera named EAB25 and EAB27 to other available ecdysteroid antisera. EAB25 had a 27-fold higher affinity for 20E than E, while EAB27 had a four-fold higher affinity for 20E. In the second part of the study, EIA protocols were developed for analyzing E and 20E produced by the mosquito Aedes aegypti. Results indicated that pelts from fourth instar larvae and ovaries from blood-fed, adult females produced E and 20E. Methanol extraction in the presence of magnesium from whole body samples altered antibody recognition of E and 20E by EIA. However, extraction with 1-butanol and two organic/water phase separations eliminated this problem and improved assay performance. We conclude the new antisera used in the EIA provide a low-cost, flexible, and sensitive method for measuring E and 20E in insects.

miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila.

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity.

Ecdysone Titer Determined by 3DE-3ß-Reductase Enhances the Immune Response in the Silkworm.

Although recent studies have demonstrated that 20-hydroxyecdysone (20E), one of the two most important hormones for development, could promote the insect innate immune response, how insects regulate 20E titer to affect the immunity after suffering pathogen attack remains unknown. In this study, to our knowledge, we first found that 20E titer was significantly elevated after bacterial infection in the domesticated silkworm, Bombyx mori. Furthermore, the elevated 20E enhanced the silkworm innate immune system against invading bacteria via ecdysone receptor. During immune response, the expression of the silkworm 3-dehydroecdysone-3ß-reductase (3DE-3ß-reductase) that converts 3DE released from prothoracic glands into ecdysone was induced. RNA interference experiments suggested that 3DE-3ß-reductase is essential to upregulate the 20E titer after bacterial infection. The rescue experiments showed that injection with the recombinant 3DE-3ß-reductase protein can significantly elevate the 20E concentration and modulate the expressions of the silkworm immune-related genes. Taken together, 20E titer determined by 3DE-3ß-reductase enhances the silkworm defense against the bacterial infection. Thus, our findings reveal an important role of the 20E synthesis pathway from 3DE in enhancing the silkworm immune response and have profound implications for the understanding of interaction mechanisms between insect hormone and immunity.

Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster.

In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. Here we provide evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. We show that early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. We also show that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down regulated and activation and translocation of transcription factor Relish is also affected.

The Y-organ secretory activity fluctuates in relation to seasons of molt and reproduction in the brachyuran crab, Metopograpsus messor (Grapsidae): Ultrastructural and immunohistochemical study.

This paper presents a first-time report on the localization, structure and seasonal secretory activity of the Y-organ of a grapsid brachyuran crab (Metopograpsus messor). Having exhibited discrete seasonality with reference to the programming of molt and reproduction, this brachyuran crab has offered us an excellent model to obtain a clear picture of the fluctuating secretory nature of the Yorgan, all the way through the reproductive (August-December) as well as the molt-reproduction active (January-May) and inactive (June-July) seasons. Ultrastructural studies revealed that the secretion of the Y-organ was at its peak in premolt crabs during molt-reproduction season (January-May). Interestingly, the Y-organs of the intermolt females that engaged in breeding activity showed higher levels of secretion than those of the molt-reproduction inactive season (June-July), implicating the gland's involvement in reproduction. Immunohistochemical studies using the antiserum raised against 2-succinyl conjugate of ecdysone have demonstrated the ecdysteroid nature of the secretion from the Y-organ, and results of the quantitative assay of ecdysteroids (through radioimmunoassay) revealed that the hormone titer fluctuates in consonance with the Y-organ's secretory activity during seasons of molt and reproduction. Pertinently, not only that the paper gives us a comprehensive understanding on the secretory activity of the Y-organ in a season-dependent fashion, it also allows us to have a better insight into the gland's function related to molting and reproduction (for the first time) in a grapsid brachyuran crab.

Bacterial, but not baculoviral infections stimulate Hemolin expression in noctuid moths.

Lepidopteran larvae are regularly infected by baculoviruses during feeding on infected plants. The differences in sensitivity to these infections can be substantial, even among closely related species. For example, the noctuids Cotton bollworm (Helicoverpa zea) and Tobacco budworm (Heliothis virescens), have a 1000-fold difference in sensitivity to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection. Recent data were interpreted to indicate that the lepidopteran immunoglobulin protein, Hemolin, is synthesized upon viral injection and therefore to participate in anti-viral responses. To investigate whether Hemolin synthesis is affected by a natural viral infection, specific transcription in fat bodies and hemocytes of H. zea and H. virescens larvae was monitored following per os infection with the baculovirus HzSNPV (H. zea single nucleopolyhedrovirus). Both moths showed the same expression pattern as seen in uninfected animals and coincided with ecdysone responses, previously known to induce Hemolin expression. In contrast, injection of lyophilized Micrococcus luteus resulted in increased Hemolin expression supporting a role for Hemolin as an immuno-responsive protein in these species. The combined data are consistent with the suggestion that while Hemolin seems to participate in the response to virus infection in the superfamily Bombycoidea, this is not true in the Noctuoidea.

Cellular immune response in Rhodnius prolixus: role of ecdysone in hemocyte phagocytosis.

In this paper we investigate in vivo and in vitro effects of orally administered azadirachtin and ecdysone on the phagocytic responses of Rhodnius prolixus 5th-instar larval hemocytes to the yeast Saccharomyces cerevisiae. Groups of insects fed non-treated blood (control) and insects that received azadirachtin plus ecdysone in the blood meal were inoculated with yeast cells in the hemocele. The injected yeast cells disappeared rapidly from the hemolymph, being removed completely by 90min after inoculation. In the insects treated only with azadirachtin the clearance of free yeast circulating particles was significantly delayed compared to the two previously mentioned groups. It was demonstrated that the binding of yeast cells to hemocytes was reduced in the insects treated only with azadirachtin in comparison to both non-treated control and azadirachtin plus ecdysone-treated groups. Phagocytosis occurred when yeast cells were added to hemocyte monolayers prepared with hemolymph from blood fed insects, treated or not with azadirachtin plus ecdysone, so that yeast cells were rapidly bound to hemocytes and internalized in high numbers. By contrast, insects treated with azadirachtin exhibited a drastic reduction in the quantity of yeast cell-hemocyte binding and subsequent internalization. In all groups, the hemocytes attached to the glass slides were predominantly plasmatocytes. The magnitude and speed of the cellular response suggests that hemocyte phagocytosis is one of the main driving forces for the clearance of free circulating yeast cells from the hemolymph. We propose that ecdysone modulates phagocytosis in R. prolixus larvae, and that this effect is antagonized by azadirachtin.

Glycogen stores in mature ovarian follicles and young embryos of Drosophila: ultrastructural changes and some biochemical correlates.

During the last phase of oogenesis in Drosophila, large amounts of carbohydrates are taken up by the oocyte and become stored in the so-called beta-spheres whose ultrastructure and histochemical properties indicate that glycogen is the predominant storage form. The ultrastructure of the beta-spheres changes at the onset of embryogenesis: they become irregular in shape and the spacing of the granular substructures (beta-particles) increases. During the first 2 h of embryonic development, the total carbohydrate content decreases sharply while at the same time the protein content increases. Presumably the carbohydrate store is used to generate energy at this phase of development. Using monoclonal antibodies against an ecdysteroid-related antigen we showed that this antigen is mostly located in the beta-spheres. The asymmetrical distribution of the antigen in the egg (more concentrated near the posterior end) correlates with the same asymmetrical distribution of the beta-spheres in the mature follicle.

Immunocytochemical localization of ecdysteroids in the life history stages of Schistosoma mansoni.

Sporocysts, cercariae and adults of S. mansoni exhibit focal immunoreactivity against anti-ecdysone serum in an indirect immunofluorescence assay. In adult males immunoreactivity is limited to cell bodies and linear connections in the parenchyma surrounding the intestinal caeca. In both unisexual and paired mature females part of the lining of the ootype is reactive, especially near the entrance of the vitelline duct; this demonstrates that females can make ecdysteroids without mal contact. Adult worms cultured completely in vitro show a similar pattern of reactivity. Immunoreactivity is strong in cercariae, but is essentially absent in miracidia.

Preparation of an ecdysone immunogen for radioimmunoassay work.

A highly improved procedure for the preparation of ecdysone-protein conjugates for immunological work is reported. Bovine thyroglobulin is succinylated and the succinylated protein is coupled to beta-ecdysone with 1-ethyl-3-(-dimethylaminopropyl)-carbodiimide in the presence of the acylation catalyst 4-dimethylaminopyridine, The antiserum obtained using this immunogen provides a radioimmunoassay sensitive to 25 pmol of beta-ecdysone. The anti-ecdysone antibody cross-reacts with alpha-ecdysone but not with cholesterol or progesterone. This procedure reverses the standard strategy for synthesizing ester linkages in hapten-protein conjugates and should have widespread applicability for the preparation of other such conjugates for immunological work.

Secretion of alpha-ecdysone by crab Y-organs in vitro.

The cultured molting glands (Y-organs) from two species of crabs secrete a product whose behavior in several chromatographic systems and whose mass spectrum are identical with those of authentic alpha-ecdysone. It appears that alpha-ecdysone is the only molting hormone secreted by the Y-organ, and that the Y-organ is the sole source of alpha-ecdysone in these animals.

[Variations in the level of ecdysones during the intermolt of male Orchestia gammarella Pallas (Crustacea. amphipoda) by radioimmunoassay]. / Etude des variations du taux des ecdysones au cours du cycle d'intermue chez le mâle d'Orchestia gammarella Pallas (Crustacé Amphipode) par dosage radioimmunologique

The molting hormone content is low during stages C, D0, D1' a-b; it rises up in D 1' c and attains a maximum in D 2. An important decrease of the hormone titer is observed at ecdysis. This titer is slightly lower at stage A and increases again at stage B. Only ecdysterone is detected at stages B and D 2.