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

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Journal of Insect Physiology 51 (2005) 27–38 www.elsevier.com/locate/jinsphys

Edysteroid receptor (EcR) shows marked differences in temporal patterns between tissues during larval-adult development in Rhodnius prolixus: correlations with haemolymph ecdysteroid titres Xanthe Vafopoulou, Colin G.H. Steel, Katherine L. Terry Biology Department, York University, 4700 Keele St., Toronto, Ontario Canada, M3J 1P3 Received 29 October 2004; accepted 1 November 2004

Abstract The presence of ecdysteroid receptor (EcR) in various tissues was studied throughout larval-adult development of the bloodsucking 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. r 2004 Elsevier Ltd. All rights reserved. Keywords: EcR; Ecdysteroids; Rhodnius; Development; Confocal microscopy; Steroid receptor

1. Introduction Circulating levels of ecdysteroids undergo systematic changes during development in all insects (Steel and Vafopoulou, 1989) that orchestrate myriad cellular changes in target cells (e.g., Riddiford, 1985; Riddiford et al., 1999). These cellular changes have been best and most fully characterized during larval-adult development when massive tissue reorganization occurs; cell multiplication, development of adult structures and Corresponding author. Tel.: +1 416 736 2100; fax: +1 416 736 5698. E-mail address: [email protected] (X. Vafopoulou).

0022-1910/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2004.11.001

autolysis of larval structures are all regulated by ecdysteroids. Cellular responses to ecdysteroids in target cells are mediated by ecdysteroid receptors (EcR). The diversity of cellular responses to ecdysteroids has been ascribed to differential expression of EcR isoforms (Bender et al., 1997; Hegstrom et al., 1998; Hodin and Riddiford, 1998; Jindra et al., 1966; Schubiger et al., 1998; Talbot et al., 1993; Truman et al., 1994). EcRs belong to the nuclear receptor superfamily and act as ligand-inducible transcription factors (see Henrich and Lepesant, 2004). Knowledge of the temporal and spatial patterns of EcR expression in insects derives almost exclusively from studies with dipterans and lepidopterans (Cho et al., 1995; Fujiwara et al., 1995; Imhof et al.,

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1993; Jindra et al., 1966; Kamimura et al., 1997; Koelle et al., 1991; Kothapalli et al., 1995; Mouillet et al., 1997; Rinehart et al., 2001; Minakuchi et al., 2002; Verras et al., 2002; Wang et al., 2002). Scant information is available regarding hemimetabolous insects (Hayward et al., 2003). The hemipteran Rhodnius prolixus has provided critical insights into the regulation of insect development since the 1930s and a massive literature exists on ecdysteroid-dependent tissue changes during development (see Wigglesworth, 1957, 1964, 1985) and on the regulation of ecdysteroid production (Vafopoulou and Steel, 2004). The enduring value of Rhodnius as an experimental insect centres on the fact that unfed larvae exist in a state of arrested development (Buxton, 1930) that is completely devoid of ecdysteroids (Steel et al., 1982). Development to the next instar can be initiated at any time by giving the insect a blood meal; ecdysteroids appear in the haemolymph within minutes of feeding (Steel et al., 1982). This system is well suited for analysis of the induction of EcR by ecdysteroids. Further, development unfolds with a remarkable temporal precision that enables accurate correlations to be made between known developmental events in tissues, ecdysteroid titres and the presence of EcR. In the present work, we examine the induction of EcR in various tissues following a blood meal and employ confocal microscopy to examine the induction of EcR in various tissues following a blood meal and to examine the relationship between EcR in various tissues and the ecdysteroid titre. We report that EcR appears and disappears at developmental times and ecdysteroid titres that vary dramatically between tissues. EcR may disappear at ecdysteroid titres higher than those that induce it. Some implications for the regulation of ecdysteroid action on target tissues are discussed.

2. Materials and methods 2.1. Animals Larvae of Rhodnius prolixus larvae were raised at 28 1C in a 12 h light: 12 h dark regime. In the present study male fifth instar larvae (last) were used. Unfed larvae exist in a state of developmental arrest; development to the adult stage is initiated by a large blood meal. Larval-adult development is counted from the day of feeding, which is designated day 0. The median day of ecdysis to the adult is day 21. Tissue dissections were carried out at various developmental times of larvaladult development; unfed state (developmentally arrested larvae), 4 h after a blood meal, day 5 (the head critical period for Rhodnius; Knobloch and Steel, 1987), day 12 (middle of development) and day 17 (advanced

development) and unfed, male adults at 7 days after ecdysis. 2.2. Antibodies The 15C mouse monoclonal Manduca EcR antibody was a generous gift from L.M. Riddiford. 9B9 and 10F1 mouse monoclonal Manduca EcR antibodies were purchased from the Developmental Studies Hybridoma Bank (University of Iowa). All these antibodies were developed against epitopes in a highly conserved region common to both A and B1 isoforms of Manduca sexta EcR (Jindra et al., 1966). 2.3. Immunoblot analysis Proteins were extracted from abdominal epidermis and fat body of day 12 scotophase larvae, unfed larvae and unfed adults. Proteins were also extracted from abdominal epidermis and fat body of unfed larvae incubated in vitro for 12 h in Rhodnius saline solution (Lane et al., 1975) containing 500 ng/ml 20E. Tissues were homogenized in 20 mM Tris-HCl (pH 7.9), 5 mM magnesium acetate, 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 0.5% Nonidet P-40, 1 mM phenylmethyl sulfonylfluoride (PNSF), 2 mg/ml leupeptin and 1 mg/ml each antipain, aprotinin and pepstatin A, followed by centrifugation at 10,000g for 15 min at 4 1C. The concentration of protein in the extract was determined by the Coommassie Brilliant Blue G-250 protein assay (BioRAD, Hercules, California). Fifteen mg of protein were separated on 10% SDSPAGE (BioRAD) and peptides were electrophoretically transferred onto nitrocellulose filters for 2 h under 200 mA. The amount of protein transferred on filters was controlled using Ponceau Red staining. The filters were blocked for 1 h in 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 1% BSA and 5% dried, non-fat milk powder. The filters were then incubated for 1 h with the 15C EcR antibody at a 1:1000 dilution. The secondary antibody was goat antimouse IgG conjugated to horseradish peroxidase at 1:200 dilution (Sigma-Aldrich, St. Louis, Missouri). Signal detection on filters was performed using the Odianisidine reaction. 2.4. Immunohistochemistry All tissues were fixed at middle-late scotophase. This consistent timing was necessary because several tissues show a striking circadian rhythm in EcR immunofluorescence and peak fluorescence occurs at this time in the 24 h cycle (unpublished observations). Tissues were fixed in freshly prepared 4% paraformaldehyde in phosphate buffered saline (PBS) (Tsang and Orchard, 1991) for 3 h. Some tissues were fixed in Bouin’s fluid overnight. Fixed

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tissues were washed thoroughly in PBS. Tissues were then preincubated in PBS containing 5% control serum and 1% Triton-X for 2 h at room temperature in order to eliminate non-specific protein binding. The primary antibody (15C EcR antibody) was used at 1:1000 dilution. Goat anti-mouse IgG conjugated to FITC (Sigma-Aldrich) was used as the secondary antibody at 1:100 dilution. Tissues were mounted in 90% glycerol in PBS in the presence of 1% DABCO (Sigma-Aldrich). Negative controls were performed by (i) replacing the primary antibody with non-immune serum (Fig. 2) and (ii) replacing the secondary antibody with PBS (not shown). The absence of detectable fluorescence in these controls demonstrated the specificity of the reaction. In order to compare the localization of EcR with chromatin within nuclei, some fixed tissues were counterstained with the nucleic acid dye propidium iodide. These tissues were treated with 100 mg/ml DNase-free RNase A (Sigma-Aldrich) in 2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) for 20 min at 37 1C prior to incubation with the primary antibody. The tissues were rinsed three times, 1 min each, in 2X SSC and then equilibrated in PBS. Following incubation with the secondary antibody, the tissues were equilibrated with 2X SSC and incubated with 500 nM propidium iodide for 5 min. Finally, the tissues were rinsed several times in 2X SSC before being mounted as above. Digital images of 1 mm optical sections were viewed using an Olympus FV300 confocal laser scanning microscope. Gain and black levels were kept constant and the pinholes were kept open by 13: Images were processed using the programs of Image J 1.27 (NIH), Corel Photo House and Power Point. The images were modified only to merge files or to adjust contrast.

3. Results All three EcR antibodies (15C, 9B9 and 10F1) produced similar results on Western blots. However, the 15C antibody was selected for all following experiments because it produced the most intense fluorescence in confocal images. Western blot analysis using the 15C EcR antibody on proteins extracted from abdominal epidermis and fat body (Fig. 1A, B) of day 12 animals in the middle of development (peak of haemolymph ecdysteroid titre; see Discussion) revealed the presence of three peptides which migrated at approximate positions of 70, 68 and 64 kDa each. These peptide bands were absent from the following two designs of control blots (1) minus primary antibody and (2) blots using irrelevant antibodies (against Bombyx prothoracicotropic hormone, bombyxin or Drosophila PERIOD protein). In blots of proteins from abdominal epidermis of unfed fifth instar larvae and unfed adults

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Fig. 1. Western blots showing immunoreactivity of peptides extracted from various tissues of Rhodnius larvae to 15C EcR antibody. Proteins in A and B were extracted from larvae in the middle of larval-adult development. A, abdominal fat body. B, abdominal epidermis. Proteins in C and D were exctracted from tissues from unfed larvae incubated in vitro with 500 ng/ml 20E for 12 h. C, abdominal fat body. D, abdominal epidermis. E and F are controls for C and D, i.e., fat body and epidermis of unfed larvae incubated without 20E. The relative molecular sizes of the peptides recognized by the antibody are shown on the left.

(which contains no ecdysteroids) these three peptides were absent (Fig. 1F). In blots of fat body proteins of unfed insects faint immunoreactivity could be detected for two of the three peptides at positions 70 and 64 kDa (Fig. 1E). A small immunoreactive peptide (about 45 kDA) in unfed fat body extract was presumed to be a break-down product because it is much smaller than any known EcR. The fact that these peptides are virtually absent from unfed animals but are prominently present in developing ones suggests that the expression of these peptides is upregulated by ecdysteroids. In order to examine this possibility, abdominal epidermis and fat body from unfed fifth instar larvae were incubated in vitro for 12 h in the presence of 500 ng/ml 20E. This concentration of 20E is about one sixth of the peak titre seen during larval-adult development (see Discussion). 20E in vitro clearly upregulated the expression of all three peptides in both fat body and epidermis (Fig. 1C,D). Since these peptides are both immunoreactive to the EcR antibody and are upregulated by physiological concentrations of 20E, they appear to represent the natural EcR of Rhodnius. EcR immunofluorescence was examined in several tissues at a number of times during larval-adult development. The tissues examined were abdominal epidermis, the medial neurosecretory cells (NSC) of the protocerebrum, PGs, premeiotic spermatocytes in cysts, abdominal fat body, the epithelium of the rectum and Malpighian tubules. The nuclei of all cell types (with one exception, see below) exhibited strong EcR fluorescence. In small cells (e.g., spermatocytes, Fig. 3) and/or at low magnifications (e.g., large neurosecretory cells, Fig. 5), the fluorescence appeared to fill the nuclei. A similar appearance was seen in image stacks of multiple consecutive 1 mm optical sections (not shown). But at higher magnifications, of single optical sections.

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(e.g., epidermis, fat body, Fig. 3) fluorescence was clearly restricted within each nucleus to an organized array of punctate spots (see Discussion). Extensive analysis of the intranuclear distribution of EcR fluorescence has been conducted and will be detailed elsewhere (Vafopoulou and Steel, in preparation). The EcR fluorescent regions are invariably located on chromatin/chromosomes. Fig. 4 shows epidermal cell nuclei at the height of secretory activity (day 12) following treatment with RNase A, then stained with anti-EcR (upper panel; green in middle panel) and finally counterstained with propidium iodide (lower panel; red in middle panel). The same general pattern of punctate spots is seen with both EcR and propidium iodide fluorescence, showing an association between EcR and chromatin. But in the merged image (middle panel), it is clear that EcR fluorescence forms a broader halo surrounding the chromatin. Further, some regions of chromatin co-localize with EcR (yellow/orange), whereas other regions of chromatin are largely devoid of EcR (red). EcR fluorescence paralleled the distribution of DNA (not RNA) in living cells (unpublished data). The localization of EcR fluorescence to chromosomes is particularly clear in known polyploid cells (Malpighian tubules, rectal epithelium, Fig. 6). The use of different fixatives (see Methods) did not significantly affect the pattern of intranuclear EcR fluorescence. These findings show that the observed punctate patterns are not fixation artefacts. The nucleoplasm of all cells was devoid of fluorescence. Similarly, no EcR fluorescence was detected in cellular compartments outside the nucleus in the present study. Therefore, the EcR antibody binds specifically to certain regions of chromatin in Rhodnius (see Discussion). Technical specificity of the antibody is illustrated in Fig. 2, which shows that nuclei emit no fluorescence when the EcR antibody is omitted; no autofluorescence was detected in any cell. In all tissues examined, nuclear EcR immunofluorescence displayed a distinctive sequence of changes during development. Further, the timing and details of this sequence varied greatly between tissues. Generally, no tissue displayed immunofluorescence in unfed fifth instar larvae or in unfed adults (Figs. 3, 4, 5 and 6). In

epidermal cells, EcR fluorescence appeared within 4 h after a blood meal and persisted throughout most of development (until at least day 17; Fig. 3, left column). The nuclei of both fat body (Fig. 3, middle column) and spermatocytes (Fig. 3, right column) also developed EcR immunofluorescence within 4 h after feeding. Individual cysts of spermatocytes showed fluorescence from either all cells within it or from none, implying that EcR fluorescence is confined to a particular stage of spermatogenesis. Nuclear EcR signal remained intense in both cell types until the middle of development. By late development, the nuclei of fat body cells had increased considerably in size and EcR fluorescence remained intense. By contrast, the signal disappeared from the nuclei of spermatocytes by day 17 (Fig. 3; compare centre and right columns). In the brain, nuclear EcR immunofluorescence was seen in numerous neurons, but was recorded primarily for the large neurosecretory cells in the dorsal protocerebrum, because these cells are individually identifiable by location, size and cell morphology (Steel and Harmsen, 1971; Morris and Steel, 1975). In these NSC, nuclear EcR immunofluorescence was absent at 4 h after feeding and the head critical period (HCP) on day 5 (Fig. 5, left column). As development advanced, nuclear signal appeared in NSC. At day 12, the nuclei of the medial NSC stained heavily. By late development (day 17) nuclear fluorescence was still present in these cells but significantly reduced. In striking contrast to other tissues, the PGs showed no EcR fluorescence at any time during development (Fig. 5, right column). In both rectal epithelial cells (Fig. 6, left column) and Malpighian tubules (Fig. 6, right column) EcR immunofluorescence was absent at 4 h after feeding. Nuclear signal became apparent by the HCP (day 5) and continued into the middle of development in both cell types. In advanced development (day 17), the rectal epithelium ceased to exhibit fluorescence, whereas the nuclei of Malpighian tubules continued to display a weakened EcR immunofluorescence. In the middle of development (day 12), several additional tissues also exhibited strong EcR signals,

Fig. 2. Control images of epidermis, brain neurons and fat body incubated with the secondary antibody but without the primary EcR antibody. Note absence of EcR fluorescence. Scale bar ¼ 10 mm.

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Fig. 3. Temporal patterns of EcR immunofluorescence in epidermis (left column), fat body (middle column) and spermatocytes (right column) during larval-adult development and in unfed adults (bottom row images). Note absence of signal in unfed larvae and unfed adults. Scale bar ¼ 15 mm.

but detailed developmental studies were not conducted. A strong nuclear signal was seen in ventral abdominal muscle, tracheal epithelial cells, testis sheath and oenocytes (Fig. 7). Fig. 7 summarizes these findings and correlates them with the titre of haemolymph ecdysteroids during larvaladult development. It is clear that EcR immunofluorescence exhibits tissue-specific temporal patterns. The time during development at which nuclear EcR fluorescence appears and subsequently disappears, as well as the duration of development for which it is present, all vary substantially between tissues. These tissue-specific EcR patterns correlate with the different levels of ecdysteroids that occur in the haemolymph during the course of development, as discussed below.

4. Discussion 4.1. Rhodnius EcR The present work employed three monoclonal antibodies prepared against the common region of the three isoforms of EcR in Manduca (Jindra et al., 1996). There are three lines of evidence indicating that these antibodies recognize the native EcR of Rhodnius. First, three immunoreactive peptides were resolved on Western blots of proteins extracted from abdominal epidermis and fat body of larvae in the middle of larvaladult development, with apparent molecular masses of 70, 68 and 64 kDa. The molecular masses of these peptides are very similar to those of other known EcRs,

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expression of EcR is induced by 20E in many insect tissues (e.g., Koelle et al., 1991; Jindra et al., 1996). Third, these three immunoreactive peptides of Rhodnius are absent in unfed larvae and adults, which lack ecdysteroids, but are present in developing larvae when the concentration of haemolymph ecdysteroids is high. Thus, these peptides are seen only in the presence of ecdysteroids in vivo. 4.2. Subcellular localization of EcR

Fig. 4. Epidermis incubated with anti-EcR (upper panel) and counterstained with the nucleic acid dye propidium iodide (lower panel). Epidermis was treated with RNase A prior to anti-EcR treatment. The middle panel shows a merged image of the upper and lower images. Green shows EcR fluorescence, red shows propidium iodide fluorescence and yellow/orange shows co-localization of green and red. Digital images represent 0.75 mm optical sections. Scale bar ¼ 10 mm.

In all the various cell types examined in this study, EcR immunofluorescence was confined to nuclei. At low magnifications, or in confocal image stacks comprising multiple 1 mm optical sections, fluorescence appeared to occupy most, if not all, of the nucleus. Such an appearance of EcR in nuclei has been reported in comparable images by other authors (e.g., Koelle et al., 1991; Hegstrom et al., 1998). But at higher magnification of individual 1mm optical sections, it was clear that nuclear EcR fluorescence was in fact highly discontinuous within all nuclei, being confined to an organized array of spots within the nucleus. Nucleoplasm showed no fluorescence. The punctate nuclear distribution of EcR fluorescence in Rhodnius cells is similar to that observed in mammalian cells, where the ligand-bound forms of many steroid hormone nuclear receptors also exhibit punctuate distribution within the nucleus (Htun et al., 1999; Baumann et al., 2001; Tomura et al., 2001). In sequential optical sections, these spots are seen to represent rod-shaped or filamentous bodies whose disposition within the nucleus varies with the cell cycle and with development. These spots invariably colocalized with spots of propidium iodide fluorescence in RAase A treated cells, demonstrating that EcR fluorescence emanated from regions of nuclear chromatin. However, not all propidium iodide fluorescent regions showed EcR fluorescence (Fig. 4), showing that EcR was not uniformly distributed throughout the chromatin. We infer that EcR fluorescence emanates from regions of chromatin with which EcR is associated in vivo. 4.3. Patterns of EcR presence during development

such as those of Manduca (Song et al., 1997), Bombyx (Swevers et al., 1995) and Chironomus (Wegmann et al., 1995). Known EcRs are expressed as 2 or 3 isoforms (see Introduction for references) or isotypes (Rauch et al., 1998). Therefore, it is highly probable that the peptides seen on Western blots represent three isoforms or isotypes of EcR in Rhodnius. The second line of evidence that these antibodies recognize the native Rhodnius EcR is that these immunoreactive peptides are upregulated by physiological concentrations of 20E in vitro in epidermis and fat body. It is well known that the

The present study also documents the temporal distribution of nuclear EcR during larval-adult development in various tissues of Rhodnius that were reported as ecdysteroid targets in the classical literature (Wigglesworth, 1957, 1964, 1985) and correlates these patterns with the known sequence of changes in the haemolymph ecdysteroid titre. Synthesis of ecdysteroids by the PGs (Vafopoulou and Steel, 1989) and their consequent appearance in the haemolymph (Steel et al., 1982) are both initiated by feeding (Fig. 8). There is a circadian rhythm in the ecdysteroid titre (Ampleford and Steel,

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Fig. 5. Developmental changes in EcR immunofluorescence in the medial NSC of the protocerebrum (left column) and PGs (right column) during larval-adult development and in young unfed adults. Note the absence of signal in PGs throughout development. In adults, the PGs are undergoing degeneration. Scale bar ¼ 10 mm.

1985; Vafopoulou and Steel, 1991) with peak values every scotophase; the data shown in Fig. 8 are photophase values and therefore are the daily minima.

No EcR signal was detected in any tissue prior to feeding; this agrees with both the absence of EcR peptides from Western blots of unfed animals and with

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Fig. 6. Developmental changes in EcR immunofluorescence in rectal epithelium (left column) and Malpighian tubules (right column) during larvaladult development and in unfed adults. Scale bar ¼ 15 mm.

the absence of ecdysteroids from the haemolymph. An EcR signal develops within 4 h of blood meal in the epidermis, fat body and spermatocytes, in synchrony

with the first small surge of ecdysteroids to about 50 ng/ ml at this time (Steel et al., 1982). This concentration of hormone is sufficient to induce transcription of EcR

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Fig. 7. EcR fluorescence in the nuclei of abdominal muscle, tracheal epithelium, testis sheath and oenocytes on day 12 (middle) of larval-adult development. Scale bar ¼ 12 mm.

mRNA in Drosophila (Karim and Thummel, 1992), Bombyx (Kamimura et al., 1997) and Manduca (Jindra et al., 1996; Hiruma et al., 1997). Therefore, the level of haemolymph ecdysteroids seen immediately after feeding is adequate to induce EcR in vivo. Further, rapid induction of EcR by 20E in vitro in Rhodnius tissues agrees with several reports of induction of EcR transcripts in tissues of various insects within 1–4 h after exposure to the hormone (Jindra et al., 1996; Kothapalli et al., 1995; Wang et al., 2002). The rapid appearance of EcR in epidermis and fat body is likely associated with the initiation of protein synthesis by 20E in these cells (Wigglesworth, 1957, 1964). The epidermal cells of the tracheae and rectum also display nuclear EcR; this is not surprising since these cells secrete a new cuticle during each moult cycle. EcR has been seen in the nuclei of Drosophila tracheal epithelium (Koelle et al., 1991), but has not been reported previously in cells of the rectum. The temporal patterns of nuclear EcR differ significantly between epidermal cells in different locations; specifically, the signal in rectal epidermis is present for only a few days during the ascending part of the titre, whereas the abdominal epidermis displays nuclear EcR throughout most of larval-adult development (Fig. 8). Therefore, epidermal cells in different locations manifest EcR at dramatically different developmental times and at different concentrations of haemolymph ecdysteroids.

The presence of EcR in the nuclei of insect spermatocytes has not been reported before. In unfed last instar larvae of Rhodnius, the development of spermatocytes is arrested in unfed larvae of Rhodnius, and is initiated promptly by a blood meal (Dumser, 1980). 20E induced mitotic divisions and maturation of sperm when injected into isolated abdomens of unfed larvae (Dumser, 1980). Therefore, the rapid appearance after feeding of EcR in spermatocytes is associated with the restoration of spermatogenesis. Although epidermis, fat body and spermatocytes all show rapid acquisition of EcR following a blood meal, it was found that EcR is lost from these cell types at different times during development and at different concentrations of haemolymph ecdysteroids. EcR remains present almost throughout development in general epidermis and fat body, but in spermatocytes EcR is lost completely while the ecdysteroid titre is still high during days 13–17 (Fig. 8). Nuclear EcR is found in numerous neurons of the dorsal protocerebrum; its presence in the brain commences around the middle of development when the ecdysteroid titre is at its peak and continues until late development during the descending part of the titre. Thus, the brain is a target of ecdysteroids in Rhodnius. The appearance of EcR in the brain coincides with the onset of anatomical reorganization of the brain e.g., differentiation of ocelli and ocellar nerves and enlargement of the compound eyes (unpublished observations).

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The medial NSC of Rhodnius show intense nuclear EcR signal during this time (Fig. 5) when they undergo considerable anatomical reorganization (Steel and Hamsen, 1971) and ultrastructural transformation (Morris and Steel, 1977). Although the nervous system in Drosophila and Manduca is a major target of ecdysteroids at metamorphosis (Truman et al., 1994; Schubiger et al., 1998), EcR has not been reported previously in NSC. Thus, the medial NSC of Rhodnius are ecdysteroid targets. This finding shows that ecdysteroids affect peptidergic neurosecretion in the brain and gives direct support to the numerous (but indirect) physiological experiments indicating a feedback regulation of the neurosecretory system by ecdysteroids in Rhodnius (Steel, 1973, 1975) and other insects (review by Sakurai, 2004). Malpighian tubules also show nuclear EcR signal throughout the main peak in the haemolymph ecdysteroid titre (Fig. 6). EcR in Malpighian tubules has also been found in Manduca (Bidmon et al., 1992) and Bombyx (Kamimura et al., 1997). The action of ecdysteroids on the Malpighian tubules of Rhodnius may be to influence fluid movement and/or ecdysteroid

catabolism as suggested for other insects (Reyrse, 1978; Rafaeli and Mordue, 1982). Several additional tissues showed nuclear EcR signal at the peak of ecdysteroid titre in the middle of development. The nuclei of ventral abdominal muscle cells exhibited strong EcR signal at this time. These cells undergo distinct differentiative changes under the influence of ecdysteroids (Wigglesworth, 1957). Degeneration and remodelling of muscle during metamorphosis in Manduca correlate with temporal expression of different EcR isoforms (Hegstrom et al., 1998). Similar phenomena may be operational in Rhodnius. The epithelial cells of the testis sheath and the oenocytes also exhibit nuclear EcR in the middle of development. The role of ecdysteroids on testis sheath is unknown. In oenocytes, the time of high secretory activity (Wigglesworth, 1970) correlates with the presence of nuclear EcR, suggesting regulation by ecdysteroids. In contrast to other tissues, the PGs of Rhodnius exhibit neither nuclear nor cytoplasmic EcR immunofluorescence at any of the times examined during larvaladult development. Even when PGs were examined at multiple time points in a 24 h cycle, no EcR signal was

Fig. 8. Correlations between haemolymph ecdysteroid titre during larval-adult development of Rhodnius (from Steel et al., 1982) and the period during development over which EcR was detected in the nuclei of various tissues (arrows at bottom).

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detected (unpublished observations). Therefore, it is unlikely that the PGs of Rhodnius are responsive to ecdysteroids. The absence of EcR in the PGs of Rhodnius implies that there is no feedback regulation of ecdysteroid synthesis in the PGs by circulating ecdysteroids. This finding is not unexpected for two reasons. First, the available evidence on feedback regulation of ecdysteroids on a developmental timescale for Rhodnius suggests that this operates via regulation of PTTH release from the brain (Steel, 1973, 1975), not via direct action on the PGs. Second, steroidogenesis by the PGs of Rhodnius is regulated by a circadian clock in the PG cells that is able to regulate both acceleration and deceleration of steroidogenesis in vitro in the absence of feedback from ecdysteroids (Vafopoulou and Steel, 1998, 2001). In conclusion, EcR in Rhodnius is found in diverse tissue types. This is consistent with the central role assigned to ecdysteroids in the orchestration of development. However, EcR first appeared in different tissues at quite different times during development and in the presence of different titres of haemolymph ecdysteroids. Equally, the disappearance of EcR occurred at different developmental times and titres in different tissues. Within a particular tissue, EcR was often seen to disappear at ecdysteroid titres well above those that induced it (Fig. 8). The mechanisms of down-regulation of EcR are not known. These complexities are not readily explained solely by the differential expression of a small number of isoforms of EcR (Henrich and Lepesant, 2004). Therefore, the present work focuses attention on the importance of tissue-specific factors (co-activators, co-repressors, chaperons and other transcriptional factors) (see Henrich and Lepesant, 2004) in the action of ecdysteroids on developing cells.

Acknowledgment Supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada. The authors gratefully acknowledge the insightful advice and suggestions of Arthur Forer, Markus Lezzi and Peter Moens. References Ampleford, E.J., Steel, C.G.H., 1985. Circadian control of a daily rhythm in haemolymph ecdysteroid titre in the insect Rhodnius prolixus (Hemiptera). General and Comparative Endocrinology 59, 453–459. Baumann, C.T., Maruvada, P., Hager, G.L., Yen, P.M., 2001. Nuclear cytoplasmic shuttling by thyroid hormone receptors: multiple protein interactions are required for nuclear retention. The Journal of Biological Chemistry 276, 11237–11245. Bender, M., Imam, F.B., Talbot, W.S., Ganetzky, B., Hogness, D.S., 1997. Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms. Cell 91, 777–788.

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Bidmon, H.J., Stumpf, W.E., Granger, N.A., 1992. Ecdysteroid receptors in the neuroendocrine–endocrine axis of a moth. Experientia 48, 42–47. Buxton, P.A., 1930. Biology of a blood sucking bug, Rhodnius prolixus. Transactions of the Entomological Society of London 78, 227–236. Cho, W.L., Kapitskaya, M.Z., Raikhel, A.S., 1995. Mosquito ecdysteroid receptor: analysis of the cDNA and expression during vitellogenesis. Insect Biochemistry and Molecular Biology 25, 19–27. Dumser, J.B., 1980. The regulation of spermatogenesis in insects. Annual Review of Entomology 25, 341–369. Fujiwara, H., Jindra, M., Newitt, R., Palli, S.R., Hiruma, K., Riddiford, L.M., 1995. Cloning of an ecdysone receptor homolog from Manduca sexta and the developmental profile of its mRNA in wings. Insect Biochemistry and Molecular Biology 25, 845–856. Hayward, D.C., Dhadialla, T.S., Zhou, S., Kuiper, M.J., Ball, E.E., Wyatt, G.R., Walker, V.K., 2003. Ligand specificity and developmental expression of RXR and ecdysone receptor in the migratory locust. Journal of Insect Physiology 49, 1135–1144. Hegstrom, C.D., Riddiford, L.M., Truman, J.W., 1998. Steroid and neuronal regulation of ecdysone receptor expression during metamorphosis in the moth, Manduca sexta. Journal of Neuroscience 18, 1786–1794. Henrich, V., Lepesant, J.-A., 2004. The ecdysteroid receptor (EcR). In: Gilbert, L.I., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science, vol 3. Elsevier Science, Oxford, in press. Hiruma, K., Bo¨cking, D., Lafont, R., Riddiford, L.M., 1997. Action of different ecdysteroids on the regulation of mRNAs for the ecdysone receptor, MHR3, dopa decarboxylase, and a larval cuticle protein in the larval epidermis of the tobacco hornworm, Manduca sexta. General and Comparative Endocrinology 107, 84–97. Hodin, J., Riddiford, L.M., 1998. The ecdysone receptor and ultraspiracle regulate the timing and progression of ovarian metamorphosis during Drosophila metamorphosis. Development, Genes and Evolution 208, 304–317. Htun, H., Holth, L.T., Walker, D., Davie, J.R., Hager, G.L., 1999. Direct visualization of the human estrogen receptor a reveals a role for ligand in the nuclear distribution of the receptor. Molecular Biology of the Cell 10, 471–486. Imhof, M.O., Rusconi, S., Lezzi, M., 1993. Cloning of a Chironomus tentans cDNA encoding a protein (cEcRH) homologous to the Drosophila melanogaster ecdysteroid receptor (dEcR). Insect Biochemistry and Molecular Biology 23, 115–124. Jindra, M., Malone, F., Hiruma, K., Riddiford, L.M., 1996. Developmental profiles and ecdysteroid regulation of the mRNA for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta. Developmental Biology 180, 258–272. Kamimura, M., Tomita, S., Kiuchi, M., Fujiwara, H., 1997. Tissuespecific and stage-specific expression of two silkworm ecdysone receptor isoforms. Ecdysteroid-dependent transcription in cultured anterior silk glands. European Journal of Biochemistry 248, 786–793. Karim, F.D., Thummel, C.S., 1992. Temporal coordination of regulatory gene expression by the steroid hormone ecdysone. EMBO Journal 11, 4083–4093. Koelle, M.R., Talbot, W.S., Segraves, W.A., Bender, M.T., Cherbas, P., Hogness, D.S., 1991. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67, 59–77. Kothapalli, R., Palli, S.R., Ladd, T.R., Sohi, S.S., Cress, D., Dhadialla, T.S., Tzertzinis, G., Retnakaran, A., 1995. Cloning and developmental expression of the ecdysone receptor gene from the spruce budworm, Choristoneura fumiferana. Developmental Genetics 17, 319–330. Knobloch, C.A., Steel, C.G.H., 1987. Effects of decapitation at the head critical period for moulting on haemolymph ecdysteroid titres

ARTICLE IN PRESS 38

X. Vafopoulou et al. / Journal of Insect Physiology 51 (2005) 27–38

in final-instar male and female Rhodnius prolixus (Hemiptera). Journal of Insect Physiology 33, 967–972. Lane, N.J., Leslie, R.A., Swales, L.S., 1975. Insect peripheral nerves: accessibility of neurohaemal regions to lanthanum. Journal of Cell Science 18, 179–197. Minakuchi, C., Nakagawa, Y., Kiuchi, M., Tomita, S., Kamimura, M., 2002. Molecular cloning, expression analysis and functional conformation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis. Insect Biochemistry and Molecular Biology 32, 999–1008. Morris, G.P., Steel, C.G.H., 1975. Ultrastructure of neurosecretory cells in the pars intercerebralis of Rhodnius prolixus (Hemiptera). Tissue and Cell 7, 73–90. Morris, G.P., Steel, C.G.H., 1977. Ultrastructural changes in the medial neurosecretory cells of Rhodnius prolixus following activation by feeding. American Zoologist 17, 929. Mouillet, J.F., Delbecque, J.P., Quennedey, B., DeLaChambre, J., 1997. Cloning of two putative ecdysteroid receptor isoforms from Tenebrio molitor and their developmental expression in the epidermis during metamorphosis. European Journal of Biochemistry 248, 856–863. Rafaeli, A., Mordue, W., 1982. The responses of the Malpighian tubules of Locusta to hormones and other stimulants. General and Comparative Endocrinology 46, 130–135. Rauch, P., Grebe, M., Elke, C., Spindler, K.-D., Spindler-Barth, M., 1998. Ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta) are phosphoproteins and are regulated differently by molting hormone. Insect Biochemistry and Molecular Biology 28, 265–275. Reyrse, J.S., 1978. Ecdysterone switches off fluid secretion at pupation in insect Malpighian tubules. Nature 271, 745–746. Riddiford, L.M., 1985. Hormone action at the cellular level. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 7. Pergaman, Oxford, pp. 37–84. Riddiford, L.M., Hiruma, K., Lan, Q., Zhou, B., 1999. Regulation and role of nuclear receptors during larval molting and metamorphosis of Lepidoptera. American Zoologist 39, 736–746. Rinehart, J.P., Cikra-Ireland, R.A., Flannagan, R.R., Denlinger, D.L., 2001. Expression of ecdysone receptor is unaffected by pupal diapause in the flesh fly, Sarcophaga crassipalpis, while its dimerization partner, USP, is downregulated. Journal of Insect Physiology 47, 915–921. Sakurai, S., 2004. Feedback Regulation. In: Gilbert, L.I., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science, vol. 3, Elsevier Science, Oxford, in press. Schubiger, M., Wade, A.A., Carney, G.E., Truman, J.W., Bender, M., 1998. Drosophila EcR-B receptor isoforms are required for larval molting and for neuron remodelling during metamorphosis. Development 125, 2053–2062. Song, Q., Alnemri, E.S., Litwack, G., Gilbert, L.I., 1997. An immunophilin is a component of the insect ecdysone receptor (EcR) complex. Insect Biochemistry and Molecular Biology 27, 973–982. Steel, C.G.H., 1973. Humoral regulation of the cerebral neurosecretory system of Rhodnius prolixus (Stal) during growth and moulting. Journal of Experimental Biology 58, 177–187. Steel, C.G.H., 1975. A neuroendocrine feedback mechanism in the insect moulting cycle. Nature 253, 267–269. Steel, C.G.H., Harmsen, R., 1971. Dynamics of the neurosecretory system in the brain of an insect, Rhodnius prolixus, during growth and moulting. General and Comparative Endocrinology 17, 125–141. Steel, C.G.H., Vafopoulou, X., 1989. Ecdysteroid titer profiles during growth and development of arthropods. In: Koolman, J. (Ed.), Ecdysone. Georg Thieme Verlag, Stuttgart, pp. 221–231. Steel, C.G.H., Bollenbacher, W.E., Smith, S.L., Gilbert, L.I., 1982. Haemolymph ecdysteroid titres during larval-adult development in

Rhodnius prolixus: correlations with molting hormone action and brain neurosecretory cell activity. Journal of Insect Physiology 28, 519–525. Swevers, L., Drevet, J.R., Lunke, M.D., Iatrou, K., 1995. The silkmoth homolog of the Drosophila ecdysone receptor (B1 isoform): cloning and analysis of expression during follicular cell differentiation. Insect Biochemistry and Molecular Biology 25, 857–866. Talbot, W.S., Swyryd, E.A., Hogness, D.S., 1993. Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell 73, 1323–1337. Tomura, A., Got, K., Morinaga, H., Nomura, M., Okabe, T., Yanase, T., Takayanagi, R., Nawata, H., 2001. The subnuclear threedimensional image analysis of androgen receptor fused to green fluorescence protein. The Journal of Biological Chemistry 276, 28395–28401. Truman, J.W., Talbot, W.S., Fahrbach, S.E., Hogness, D.S., 1994. Ecdysone receptor expression in the CNS correlates with stagespecific responses to ecdysteroids during Drosophila and Manduca development. Development 120, 219–234. Tsang, P.W., Orchard, I., 1991. Distribution of FMRF-amide-related peptides in the blood feeding bug Rhodnius prolixus. Journal of Comparative Neurology 311, 17–32. Vafopoulou, X., Steel, C.G.H., 1989. Developmental and diurnal changes in ecdysteroid biosynthesis by prothoracic glands of Rhodnius prolixus (Hemiptera) in vitro during the last larval instar. General and Comparative Endocrinology 74, 484–493. Vafopoulou, X., Steel, C.G.H., 1991. Circadian regulation of synthesis of ecdysteroids by prothoracic glands of the insect Rhodnius prolixus: evidence of a dual oscillator system. General and Comparative Endocrinology 74, 484–493. Vafopoulou, X., Steel, C.G.H., 1998. A photosensitive circadian oscillator in an insect endocrine gland: photic induction of rhythmic steroidogenesis in vitro. Journal of Comparative Physiology A 182, 343–349. Vafopoulou, X., Steel, C.G.H., 2001. Induction of rhythmicity in prothoracicotropic hormone and ecdysteroids in Rhodnius prolixus: roles of photic and neuroendocrine Zeitgebers. Journal of Insect Physiology 47, 935–941. Vafopoulou, X., Steel, C.G.H., 2004. Circadian organization of the endocrine system. In: Gilbert, L.I., Iatrou, K., Gill, S. (Eds.), Comprehensive Molecular Insect Science, vol. 3, Elsevier Science, Oxford, in press. Verras, M., Gourzi, P., Zacharopoulou, A., Mintzas, A.C., 2002. Developmental profiles and ecdysone regulation of the mRNAs for two ecdysone receptor isoforms in the Mediterranean fruit fly Ceratitis capitata. Insect Molecular Biology 11, 553–565. Wang, S.F., Li, C., Sun, G., Zhu, J., Raikhel, S., 2002. Differential expression and regulation by 20-hydroxyecdysone of mosquito ecdysteroid receptor isoforms A and B. Molecular and Cellular Endocrinology 196, 29–42. Wegmann, I.S., Quack, S., Spindler, K.D., Dorsch-Ha¨sler, K., Vo¨gtli, M., Lezzi, M., 1995. Immunological studies on the developmental and chromosomal distribution of ecdysteroid receptor protein in Chironomus tentans. Archives of Insect Biochemistry and Physiology 30, 95–114. Wigglesworth, V.B., 1957. The action of growth hormones in insects. Symposia of the Society for Experimental Biology 11, 204–227. Wigglesworth, V.B., 1964. The hormonal regulation of growth and reproduction in insects. Advances in Insect Physiology 2, 247–336. Wigglesworth, V.B., 1970. Structural lipids in the insect cuticle and the function of the oenocytes. Tissue and Cell 2, 155–179. Wigglesworth, V.B., 1985. Historical perspectives. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 7. Pergamon Press, Oxford, pp. 1–24.