Halloween genes are expressed with a circadian rhythm during development in prothoracic glands of the insect RHODNIUS PROLIXUS.

We analyse the developmental and circadian profiles of expression of the genes responsible for ecdysteroidogenesis (Halloween genes) in the PGs of Rhodnius prolixus throughout larval-adult development. Extensive use of in vitro techniques enabled multiple different parameters to be measured in individual PGs. Expression of disembodied and spook closely paralleled the ecdysteroid synthesis of the same PGs, and the ecdysteroid titre in vivo, but with functionally significant exceptions. Various tissues other than PGs expressed one, both or neither genes. Both gonads express both genes in pharate adults (larvae close to ecdysis). Both genes were expressed at low, but significant, levels in UF Rhodnius, raising questions concerning how developmental arrest is maintained in UF animals. IHC confirmed the subcellular localisation of the coded proteins. Gene knockdown suppressed transcription of both genes and ecdysteroid synthesis, with spook apparently regulating the downstream gene disembodied. Transcription of both genes occurred with a daily rhythm (with peaks at night) that was confirmed to be under circadian control using aperiodic conditions. The complex behaviour of the rhythm in LL implied two anatomically distinct oscillators regulate this transcription rhythm. First, the circadian clock in the PGs and second, the circadian rhythm of of Rhodnius PTTH which is released rhythmically from the brain under control of the circadian clock therein, both of which were described previously. We conclude ecdysteroidogenesis in Rhodnius PGs employs a similar pathway as other insects, but its control is complex, involving mechanisms both within and outside the PGs.

The prothoracicotropic hormone (PTTH) of Rhodnius prolixus (Hemiptera) is noggin-like: Molecular characterisation, functional analysis and evolutionary implications.

Prothoracicotropic hormone (PTTH) is a central regulator of insect development that regulates the production of the steroid moulting hormones (ecdysteroids) from the prothoracic glands (PGs). Rhodnius PTTH was the first brain neurohormone discovered in any animal almost 100 years ago but has eluded identification and no homologue of Bombyx mori PTTH occurs in its genome. Here, we report Rhodnius PTTH is the first noggin-like PTTH found. It differs in important respects from known PTTHs and is the first PTTH from the Hemimetabola (Exopterygota) to be fully analysed. Recorded PTTHs are widespread in Holometabola but close to absent in hemimetabolous orders. We concluded Rhodnius PTTH likely differed substantially from the known ones. We identified one Rhodnius gene that coded a noggin-like protein (as defined by Molina et al., 2009) that had extensive similarities with known PTTHs but also had two additional cysteines. Sequence and structural analysis showed known PTTHs are closely related to noggin-like proteins, as both possess a growth factor cystine knot preceded by a potential cleavage site. The gene is significantly expressed only in the brain, in a few cells of the dorsal protocerebrum. We vector-expressed the sequence from the potential cleavage site to the C-terminus. This protein was strongly steroidogenic on PGs in vitro. An antiserum to the protein removed the steroidogenic protein released by the brain. RNAi performed on brains in vitro showed profound suppression of transcription of the gene and of production and release of PTTH and thus of ecdysteroid production by PGs. In vivo, the gene is expressed throughout development, in close synchrony with PTTH release, ecdysteroid production by PGs and the ecdysteroid titre. The Rhodnius PTTH monomer is 17kDa and immunoreactive to anti-PTTH of Bombyx mori (a holometabolan). Bombyx PTTH also mildly stimulated Rhodnius PGs. The two additional cysteines form a disulfide at the tip of finger 2, causing a loop of residues to protrude from the finger. A PTTH variant without this loop failed to stimulate PGs, showing the loop is essential for PTTH activity. It is considered that PTTHs of Holometabola evolved from a noggin-like protein in the ancestor of Holometabola and Hemiptera, c.400ma, explaining the absence of holometabolous-type PTTHs from hemimetabolous orders and the differences of Rhodnius PTTH from them. Noggin-like proteins studied from Hemiptera to Arachnida were homologous with Rhodnius PTTH and may be common as PTTHs or other hormones in lower insects.

Neuropeptide- and serotonin- cells in the brain of Rhodnius prolixus (Hemiptera) associated with the circadian clock.

The neuronal pathways of the circadian clock in the brain of R. prolixus have been described in detail previously, but there is no information concerning the cells or their pathways which relay either inputs to the clock (e.g. for light entrainment), or outputs from it to driven rhythms. Here, we employ antisera to three neuropeptides (type A allatostatin-7, crustacean cardioactive peptide and FMRFamide), and serotonin in confocal laser scanning immunohistochemistry to analyze the distribution of cell bodies and their projections in relation to the principle circadian clock cells (lateral cells, LNs) for all four neuron types. LNs are revealed following labelling with anti- pigment dispersing factor in double labelled preparations. Regions of potential communication between ramifications of the LNs and each of the four other neuron types is described (identified by close superposition of their neurites in various brain regions), as is their detailed projections within the brain. Neuromodulation is sometimes suggested by close, but not intimate, proximity of varicosities of neurites. We infer that some neuron types comprise input pathways to the LNs, some are outputs to neuroendocrine or behavioral rhythms, and others participate in both input and output pathways, sometimes by the same neuron type but in different locations. For example, one retinula cell in each ommatidium is immunoreactive for allatostatin A; its axon projects to the medulla making superpositions with LNs, as do serotonin cells in the optic lobe, indicating roles of both neuron types in light input (entrainment) to the clock. But in other brain areas, these same types appear to mediate outputs from the clock. The accessory medulla has been widely reported as the principle center of integration in other insects; but we found sparse evidence of this in R. prolixus as it contains few neurites other than those from the clock cells. Rather, the importance of neural pathways involving the medulla and the superior protocerebrum is emphasized. We conclude that there is a vast and complex web of interactions in the brain with the LNs, which potentially receive multiple pathways of inputs and outputs that could drive rhythmicity in a multitude of downstream cells, rendering a host of output pathways rhythmic, notably hormone release from neurosecretory cells and behaviors.

Mitochondria and the insect steroid hormone receptor (EcR): A complex relationship.

The actions of the insect steroid molting hormones, ecdysteroids, on the genome of target cells has been well studied, but little is known of their extranuclear actions. We previously showed in Rhodnius prolixus that much of the ecdysteroid receptor (EcR) resides in the cytoplasm of various cell types and undergoes shuttling between nucleus and cytoplasm with circadian periodicity, possibly using microtubules as tracks for translocation to the nucleus. Here we report that cytoplasmic EcR appears to be also involved in extranuclear actions of ecdysteroids by association with the mitochondria. Western blots of subcellular fractions of brain lysates revealed that EcR is localized in the mitochondrial fraction, indicating an intimate association of EcR with mitochondria. Confocal laser microscopy and immunohistochemistry using anti-EcR revealed abundant co-localization of EcR with mitochondria in brain neurons and their axons, especially intense in the subplasmalemmal region, raising the possibility of EcR involvement in mitochondrial functions in subplasmalemmal microdomains. When mitochondria are dispersed by disruption of microtubules with colchicine, EcR remains associated with mitochondria showing strong receptor association with mitochondria. Treatment in vitro with ecdysteroids of brains of developmentally arrested R. prolixus (containing neither ecdysteroids nor EcR) induces EcR and abundant co-localization with mitochondria in neurons, concurrently with a sharp increase of the mitochondrial protein COX 1, suggesting involvement of EcR in mitochondrial function. These findings align EcR with various vertebrate steroid receptors, where actions of steroid receptors on mitochondria are widely known and suggest that steroid receptors across distant phyla share similar functional attributes.

Synergistic induction of the clock protein PERIOD by insulin-like peptide and prothoracicotropic hormone in Rhodnius prolixus (Hemiptera): implications for convergence of hormone signaling pathways.

We showed previously that release of the cerebral neurohormones, bombyxin (an insulin-like peptide, ILP) and prothoracicotropic hormone (PTTH) from the brain have strong circadian rhythms, driven by master clock cells in the brain. These neurohormone rhythms synchronize the photosensitive brain clock with the photosensitive peripheral clock in the cells of the prothoracic glands (PGs), in which both regulate steroidogenesis. Here, using immunohistochemistry and confocal laser scanning microscopy, we show these neurohormones likely act on clock cells in the brain and PGs by regulating expression of PERIOD (PER) protein. PER is severely reduced in the nuclei of all clock cells in continuous light, but on transfer of tissues to darkness in vitro, it is rapidly induced. A 4h pulse of either PTTH or ILPs to brain and PGs in vitro both rapidly and highly significantly induce PER in the nuclei of clock cells. Administration of both neurohormones together induces more PER than does either alone and even more than does transfer to darkness, at least in PG cells. These are clearly non-steroidogenic actions of these peptides. In the peripheral oscillators salivary gland (SG) and fat body cells, neither bombyxin nor PTTH nor darkness induced PER, but a combination of both bombyxin and PTTH induced PER. Thus, PTTH and ILPs exert synergistic actions on induction of PER in both clock cells and peripheral oscillators, implying their signaling pathways converge, but in different ways in different cell types. We infer clock cells are able to integrate light cycle information with internal signals from hormones.

Insulin-like and testis ecdysiotropin neuropeptides are regulated by the circadian timing system in the brain during larval-adult development in the insect Rhodnius prolixus (Hemiptera).

Insulin-like peptides (ILPs) regulate numerous functions in insects including growth, development, carbohydrate metabolism and female reproduction. This paper reports the immunohistochemical localization of ILPs in brain neurons of Rhodnius prolixus and their intimate associations with the brain circadian clock system. In larvae, three groups of neurons in the protocerebrum are ILP-positive, and testis ecdysiotropin (TE) is co-localized in two of them. During adult development, the number of ILP groups increased to four. A blood meal initiates transport and release of ILPs, indicating that release is nutrient dependent. Both production and axonal transport of ILPs continue during adult development with clear cytological evidence of a daily rhythm that closely correlates with the daily rhythm of ILPs release from brains in vitro. The same phenomena were observed with TE previously. Double labeling for ILPs and pigment dispersing factor (PDF) (contained in the brain lateral clock cells, LNs) revealed intimate associations between axons of the ILP/TE cells and PDF-positive axons in both central brain and retrocerebral complex, revealing potential neuronal pathways for circadian regulation of ILPs and TE. Similar close associations were found previously between LN axons and axons of the brain neurons producing the neuropeptide prothoracicotropic hormone. Thus, the brain clock system controls rhythmicity in multiple brain neurohormones. It is suggested that rhythms in circulating ILPs and TE act in concert with known rhythms of circulating ecdysteroids in both larvae and adults to orchestrate the timing of cellular responses in diverse tissues of the animal, thereby generating internal temporal order within it.

Cytoplasmic travels of the ecdysteroid receptor in target cells: pathways for both genomic and non-genomic actions.

Signal transduction of the insect steroid hormones, ecdysteroids, is mediated by the ecdysteroid receptor, EcR. In various cells of the insect Rhodnius prolixus, EcR is present in both the nucleus and the cytoplasm, where it undergoes daily cycling in abundance and cellular location at particular developmental times of the last larval instar that are specific to different cell types. EcR favors a cytoplasmic location in the day and a nuclear location in the night. This study is the first to examine the potential mechanisms of intracellular transport of EcR and reveals close similarities with some of its mammalian counterparts. In double and triple labels using several antibodies, immunohistochemistry, and confocal laser scanning microscopy, we observed co-localization of EcR with the microtubules (MTs). Treatments with either the MT-stabilizing agent taxol or with colchicine, which depolymerizes MTs, resulted in considerable reduction in nuclear EcR with a concomitant increase in cytoplasmic EcR suggesting that MT disruption inhibits receptor accumulation in the nucleus. EcR also co-localizes with the chaperone Hsp90, the immunophilin FKBP52, and the light chain 1 of the motor protein dynein. All these factors also co-localize with MTs. We propose that in Rhodnius, EcR exerts its genomic effects by forming a complex with Hsp90 and FKBP52, which uses dynein on MTs as a mechanism for daily nucleocytoplasmic shuttling. The complex is transported intact to the nucleus and dissociates within it. We propose that EcR utilizes the cytoskeletal tracks for movement in a manner closely similar to that used by the glucocorticoid receptor. We also observed co-localization of EcR with mitochondria which suggests that EcR, like its mammalian counterparts, may be involved in the coordination of non-genomic responses of ecdysteroids in mitochondria.

Metamorphosis of a clock: remodeling of the circadian timing system in the brain of Rhodnius prolixus (Hemiptera) during larval-adult development.

The rhythmic phenomena expressed by organisms change over their lifetimes, but little is known of accompanying reorganization of the central circadian timing system in the brain. Especially dramatic changes in overt rhythms and morphology occur during transformation of larval insects into the adult form (metamorphosis). In Rhodnius prolixus, both the physiology of metamorphosis and its hormonal control are known in detail. Here we report changes in the brain timing system as revealed by pigment dispersing factor immunohistochemistry and confocal microscopy. Most of the features of the larval system are retained, but new clock cells differentiate and the arborizations of their axons increase in complexity, as do pathways connecting the lateral (LNs) and dorsal (DNs) groups of clock neurons. Early in metamorphosis, the LNs increase from 8 to 11 in number, becoming five small and six large LNs. Two large LNs then migrate to new positions in the protocerebrum. Another clock cell differentiates in the posterior protocerebrum. Each change occurs at a characteristic concentration of the ecdysteroid molting hormones that regulate metamorphosis. Clock cell axons invade the mushroom body and corpus allatum and travel down the ventral nerve cord. New overt rhythms develop during metamorphosis, in which these structures participate. The neuroendocrine cells of the brain receive more extensive branches of clock cell axons than in larvae. These increases in size and complexity of the circadian system during metamorphosis imply a greater complexity and diversity of outputs from it to both behavioral and hormonal rhythms in the adult.

Rhythmic release of prothoracicotropic hormone from the brain of an adult insect during egg development.

Prothoracicotropic hormone (PTTH) is a brain neurohormone that has been studied for over 80 years. The only known target of PTTH is the prothoracic glands (PGs) of larvae, which synthesize the insect molting hormones (ecdysteroids) and a massive literature exists on this axis. The PGs degenerate around the time of adult emergence, yet presence of PTTH has been reported in the brains of several adult insects. Using an in vitro bioassay system, we confirm that PTTH is present in the adult female brain of Rhodnius prolixus. The material is electrophoretically, immunologically and biologically indistinguishable from larval PTTH. The amount of PTTH in the brain shows a daily rhythm during egg development. We show that brains in vitro release PTTH with a daily rhythm over this period of time. PTTH is released at each scotophase. This is the first report that PTTH is released from the adult brain and functions as a hormone, inviting explanation of its function. Larval PTTH is also known to be released with a daily rhythm, and the clock in the brain controls both larval and adult rhythms. The potential significance of rhythmic PTTH release in female adults is discussed in relation to the regulation of ecdysteroids, egg development and the concept of internal temporal order.

The circadian timing system in the brain of the fifth larval instar of Rhodnius prolixus (hemiptera).

The brain of larval Rhodnius prolixus releases neurohormones with a circadian rhythm, indicating that a clock system exists in the larval brain. Larvae also possess a circadian locomotor rhythm. The present paper is a detailed analysis of the distribution and axonal projections of circadian clock cells in the brain of the fifth larval instar. Clock cells are identified as neurons that exhibit circadian cycling of both PER and TIM proteins. A group of eight lateral clock neurons (LNs) in the proximal optic lobe also contain pigment-dispersing factor (PDF) throughout their axons, enabling their detailed projections to be traced. LNs project to the accessory medulla and thence laterally toward the compound eye and medially into a massive area of arborizations in the anterior protocerebrum. Fine branches radiate from this area to most of the protocerebrum. A second group of clock cells (dorsal neurons [DNs]), situated in the posterior dorsal protocerebrum, are devoid of PDF. The DNs receive two fine axons from the LNs, indicating that clock cells throughout the brain are integrated into a timing network. Two axons of the LNs cross the midline, presumably coordinating the clock networks of left and right sides. The neuroarchitecture of this timing system is much more elaborate than any previously described for a larval insect and is very similar to those described in adult insects. This is the first report that an insect timing system regulates rhythmicity in both the endocrine system and behavior, implying extensive functional parallels with the mammalian suprachiasmatic nucleus.

Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera).

We have shown previously that EcR in larval Rhodnius is present in the cytoplasm of various cell types and undergoes daily cycling in abundance in the cytoplasm (Vafopoulou and Steel, 2006. Cell Tissue Res 323:443-455). It is unknown which organelles are associated with EcR. Here, we report that cytoplasmic EcR in prothoracic gland cells is associated with both microtubules and mitochondria, and discuss the implications for both nuclear and non-genomic actions of EcR. EcR was localized immunohistochemically using several antibodies to EcR of Manduca and Drosophila and a confocal laser scanning microscope. Double labels were made to visualize EcR and (1) microtubules (using an antibody to tyrosylated alpha-tubulin) and (2) mitochondria (using a fluorescent MitoTracker probe), both after stabilization of microtubules with taxol. EcR co-localized with both tubulin and mitochondria. All the different EcR antibodies produced similar co-localization patterns. EcR was seen in the perinuclear aggregation of mitochondria, indicating that mitochondria are targets of ecdysone, which could influence mitochondrial gene transcription. EcR was also distributed throughout the microtubule network. Co-localization of EcR with tubulin or mitochondria was maintained after depolymerization of microtubules with colchicine. Treatment with taxol resulted in accumulation of EcR in the cytoplasm and simultaneous depletion of EcR from the nucleus, suggesting that microtubules may be involved in targeted intracellular transport of EcR to the nucleus (genomic action) or may play a role in rapid ecdysone signal transduction in the extranuclear compartment, i.e., in non-genomic actions of ecdysone. These findings align EcR more closely with steroid hormone receptors in vertebrates.

Neuroanatomical relations of prothoracicotropic hormone neurons with the circadian timekeeping system in the brain of larval and adult Rhodnius prolixus (Hemiptera).

This paper reports the localization in the Rhodnius prolixus brain of neurons producing the key neuropeptide that regulates insect development, prothoracicotropic hormone (PTTH) and describes intimate associations of the PTTH neurons with the brain circadian timekeeping system. Immunohistochemistry and confocal laser scanning microscopy revealed that the PTTH-positive neurons in larvae are located in a single group in the lateral protocerebrum. Their number increases from two in the last larval instar to five during larval-adult development. In adults, there are two distinct groups of these neurons composed of two cells each. A daily rhythm in content of PTTH-positive material occurs in both the somata and the axons in both larval and adult stages. These rhythms correlate with previous evidence of a circadian rhythm of PTTH release from brains in vitro. The key circadian clock cells of Rhodnius are eight neurons, which co-express pigment-dispersing factor (PDF) and the canonical clock proteins PER and TIM; PDF fills the axons. Equivalent cells control behavioral rhythms in other insects. Double labeling revealed intimate associations between axons of larval PTTH neurons and clock neurons, indicating a neuronal pathway from the brain timekeeping system for circadian control of PTTH release. Additional PDF neurons appear in the adult, associated with the second group of PTTH neurons. These findings provide the first direct evidence that neurons of the insect brain timekeeping system control hormone rhythms. The range of functions regulated by this timekeeping system is quite similar to those of the vertebrate suprachiasmatic nucleus, for which the insect system is a valuable model.

Spatial and temporal distribution of the ecdysteroid receptor (EcR) in haemocytes and epidermal cells during wound healing in the crayfish, Procambarus clarkii.

Wound healing in crustaceans preserves the integrity of the integument and prevents entry of pathogens. We studied the interaction between the moulting hormones (ecdysteroids) and the cellular events under the wound during wound healing with or without bacteria infection. Wounding of the carapace by abrasion induced a rapid increase in circulating ecdysteroid levels to a low sustained plateau level for about 12 days, followed by a sharp premoult peak and moulting. Within 48h of wounding, the nuclear receptor for ecdysteroids (EcR) appeared in the nuclei of haemocytes (hyaline, semigranular and granulocytes), visualized by confocal laser scanning microscopy and anti-EcR. Hyaline haematocytes aggregated in layers below the wound site and granulocytes engaged in phagocytosis. Therefore, the immune system responds directly and rapidly to ecdysteroids. Epidermal cells developed EcR only several days after the haemocytes and only under intact carapace, not under the wound where they appeared apoptotic. At the wound margin, EcR-positive epidermal cells and fibroblasts proceeded to migrate across the wound between the layers of haemocytes. Epidermis was fully regenerated by day 15; at this time the ecdysteroid titre began rising towards a premoult peak and EcR disappeared from the nuclei of epidermal cells suggesting that high amounts of ecdysteroids exert negative control on EcR. When bacteria were injected at the time of wounding, both the plateau level of ecdysteroid titre and the cellular events of wound healing were prolonged by 5-7 days, showing that healing of the wound is slower and that the duration of the plateau phase of the titre depends on the degree of assault on the animal. We conclude that the low levels of ecdysteroids induced by wounding activate the immune system to begin healing below the wound and also stimulate adjacent epidermal cells to commence the process of wound repair.

Circadian orchestration of developmental hormones in the insect, Rhodnius prolixus.

This review presents a new perspective on the circadian regulation and functions of insect developmental hormones. In Rhodnius prolixus (Hemiptera), the brain neuropeptide prothoracicotropic hormone (PTTH) is released with a circadian rhythm that is controlled by paired photosensitive clocks in the brain. These clocks comprise the dorsal and lateral PER/TIM clock neurons known to regulate behavioral rhythms in Drosophila. Axons of PTTH and clock cells make close contact. Photosensitive PER/TIM clocks also reside in the paired prothoracic glands (PGs), which generate rhythmic synthesis and release of the ecdysteroid molting hormones. The PG clocks are entrained by both light and PTTH. These four clocks are coupled together by both nerves and hormones into a timing system whose primary regulated output is the circadian rhythm of ecdysteroids in the hemolymph. This complex timing system appears necessary to ensure circadian organization of the gene expression that is induced in target cells by ecdysteroids via circadian cycling of the nuclear ecdysteroid receptor (EcR). This multioscillator system serves to transduce 'the day outside' into endocrine rhythms that orchestrate 'the day inside'. It has many functional similarities with vertebrate circadian systems.

Non-genomic ecdysone effects and the invertebrate nuclear steroid hormone receptor EcR--new role for an "old" receptor?

The ecdysteroids (Ec), invertebrate steroid hormones, elicit genomic but also non-genomic effects. By analogy to vertebrates, non-genomic responses towards Ec may be mediated not only by distinct membrane-integrated but also by membrane-associated receptors like the classical nuclear ecdysteroid receptor (EcR) of arthropods. This is supported by a comparison of physiological properties between invertebrate and vertebrate steroid hormone systems and recent findings on the subcellular localization of EcR. The measured or predicted high degree of conformational flexibility of both Ec and the ligand binding domain (LBD) of EcR give rise to a conformational compatibility model: the compatibility between conformations of the cognate receptor's ligand binding domain and structures or conformations of the ligand would determine their interaction and eventually the initiation of genomic versus non-genomic pathways. This model could also explain why specific non-genomic effects are generally not observed with non-steroidal agonists of the bisacylhydrazine group.

Ecdysteroid hormone nuclear receptor (EcR) exhibits circadian cycling in certain tissues, but not others, during development in Rhodnius prolixus (Hemiptera).

The insect moulting hormones, viz. the ecdysteroids, regulate gene expression during development by binding to an intracellular protein, the ecdysteroid receptor (EcR). In the insect Rhodnius prolixus, circulating levels of ecdysteroids exhibit a robust circadian rhythm. This paper demonstrates associated circadian rhythms in the abundance and distribution of EcR in several major target tissues of ecdysteroids, but not in others. Quantitative analysis of immunofluorescence images obtained by confocal laser-scanning microscopy following the use of anti-EcR has revealed a marked daily rhythm in the nuclear abundance of EcR in cells of the abdominal epidermis, brain, fat body, oenocytes and rectal epithelium of Rhodnius. This EcR rhythm is synchronous with the rhythm of circulating hormone levels. It free-runs in continuous darkness for several cycles, showing that EcR nuclear abundance is under circadian control. Circadian control of a nuclear receptor has not been shown previously in any animal. We infer that the above cell types detect and respond to the temporal signals in the rhythmic ecdysteroid titre. In several cell types, the rhythm in cytoplasmic EcR peaks several hours prior to the EcR peak in the nucleus each day, thereby implying a daily migration of EcR from the cytoplasm to the nucleus. This finding shows that EcR is not a constitutive nuclear receptor, as has previously been assumed. In the brain, rhythmic nuclear EcR has been found in peptidergic neurosecretory cells, indicating a potential pathway for feedback regulation of the neuroendocrine system by ecdysteroids, and also in regions containing circadian clock neurons, suggesting that the circadian timing system in the brain is also sensitive to rhythmic ecdysteroid signals.

Testis ecdysiotropic peptides in Rhodnius prolixus: biological activity and distribution in the nervous system and testis.

In Rhodnius prolixus, testes from both pharate adult and adult males are shown to produce and release ecdysteroids in vitro. Proteinaceous brain extracts from these stages caused stimulation of ecdysteroid production by testes of unfed adults. Therefore, the brain of Rhodnius contains peptides with testis ecdysiotropic activity. The Lymantria testis ecdysiotropin (LTE) also stimulated the in vitro production of ecdysteroids by unfed adult testis but had no stimulatory effect on prothoracic glands. Western blot analysis of brain peptides using anti-LTE revealed the presence of several medium to small size immunoreactive peptides. Two of these peptides with sizes of 16.8 and 11.0 kDa were present only during pharate adult development and the adult stage. Immunohistochemical analysis using confocal laser scanning microscopy revealed abundant LTE-immunoreactive material in cytoplasmic granules of specific neurosecretory cells in the brain and suboesophageal ganglion and the epithelium of the testis sheath. Clusters of two cytologically distinct cell types were seen within the medial neurosecretory cells (MNC) and also a pair of neurons in the posterior protocerebrum. Feeding in both larvae and adult males resulted in massive release of LTE-immunoreactive material from the MNC cells, suggesting a role of LTE-related peptides in both larval-adult development and in male reproductive development. Release from the MNC cells of LTE-immunoreactive material exhibited a clear daily cycling during larval-adult development, which was synchronous with the rhythms of release of prothoracicotropic hormone and bombyxin reported previously. The testis sheath exhibited intense immunofluorescence in pharate adults and unfed adults, which disappeared following a blood meal. It is concluded that LTE-related peptides are developmentally regulated in several locations and may act as ecdysiotropins in Rhodnius. Those in the MNC cells are very probably classical hormones, i.e. are transported to their target sites via the insect haemolymph.

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.