Ecdysteroidogenesis in Heliothis virescens (Lepidoptera: Noctuidae): Recombinant Prothoracicotropic Hormone and Brain Extract Show Comparable Effects.

Prothoracicotropic hormone (PTTH) is a neuropeptide that triggers a cascade of events within the prothoracic gland (PG) cells, leading to the activation of all the crucial enzymes involved in ecdysone biosynthesis, the main insect steroid hormone. Studies concerning ecdysteroidogenesis predicted PTTH action using brain extract (BE), consisting in a complex mixture in which some components positively or negatively interfere with PTTH-stimulated ecdysteroidogenesis. Consequently, the integration of these opposing factors in steroidogenic tissues leads to a complex secretory pattern. A recombinant form of prothoracicotropic hormone (rPTTH) from the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) was expressed and purified to perform in vitro tests in a standard and repeatable manner. A characterization of rPTTH primary and secondary structures was performed. The ability of rPTTH and H. virescens BE to stimulate ecdysteroidogenesis was investigated on the third day of fifth larval stage. rPTTH activity was compared with the BE mixture by enzyme immunoassay and western blot, revealing that they equally stimulate the production of significant amount of ecdysone, through a transduction cascade that includes the TOR pathway, by the phosphorylation of 4E binding protein (4E-BP) and S6 kinase (S6K), the main targets of TOR protein. The results of these experiments suggest the importance of obtaining a functional pure hormone to perform further studies, not depending on the crude brain extract, composed by different elements and susceptible to different uncontrollable variables.

Royal jelly attenuates cadmium-induced nephrotoxicity in male mice.

Cadmium exposure induces nephrotoxicity by mediating oxidative stress, inflammation, and apoptosis. The purpose of this study was to examine the protective effect of royal jelly on Cd-induced nephrotoxicity. Adult male mice were distributed randomly into 4 clusters: untreated, royal jelly-treated (85 mg/kg, oral), CdCl2-treated (6.5 mg/kg, intraperitoneal), and pretreated with royal jelly (85 mg/kg) 2 h before CdCl2 injection (6.5 mg/kg, intraperitoneal) for seven consecutive days. Cd concentration in the renal tissue and absolute kidney weight of the Cd-treated mice were significantly higher than those of control group. The levels of kidney function markers, kidney injury molecules-1 (KIM-1), metallothionein, lipid peroxidation, nitric oxide, tumor necrosis factor-α, interleukin-1ß, and the apoptosis regulators Bax and caspases-3 also increased significantly in the renal tissue of Cd-treated mice, whereas the levels of glutathione, antioxidant enzyme activities, and the apoptosis inhibitor Bcl-2 were significantly reduced in the renal tissue of Cd-treated group. Histopathological studies showed vacuolation and congested glomeruli in the kidney tissue of Cd-treated mice. However, all aforementioned Cd-induced changes were attenuated by pretreatment with royal jelly. We therefore concluded that royal jelly attenuated Cd-induced nephrotoxicity and it is suggested that this nephroprotective effect could be linked to its ability to promote the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) pathway.

Role of protein phosphatase 2A in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori.

In the present study, the roles of a major serine/threonine protein phosphatase 2A (PP2A) in prothoracicotropic hormone (PTTH)-stimulated prothoracic glands (PGs) of Bombyx mori were evaluated. Immunoblotting analysis showed that Bombyx PGs contained a structural A subunit (A), a regulatory B subunit (B), and a catalytic C subunit (C), with each subunit undergoing development-specific changes. The protein levels of each subunit were not affected by PTTH treatment. However, the highly conserved tyrosine dephosphorylation of PP2A C subunit (PP2Ac), which appears to be related to activity, was increased by PTTH treatment in a time-dependent manner. We further demonstrated that phospholipase C (PLC), Ca2+, and reactive oxygen species (ROS) are upstream signaling for the PTTH-stimulated dephosphorylation of PP2Ac. The determination of PP2A enzymatic activity showed that PP2A enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Okadaic acid (OA), a specific PP2A inhibitor, prevented the PTTH-stimulated dephosphorylation of PP2Ac and reduced both basal and PTTH-stimulated PP2A enzymatic activity. The determination of ecdysteroid secretion showed that treatment with OA did not affect basal ecdysteroid secretion but did significantly inhibit PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PP2A activity is involved in ecdysteroidogenesis. Treatment with OA stimulated the basal phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) without affecting PTTH-stimulated ERK and 4E-BP phosphorylation. From these results, we hypothesize that PTTH-regulated PP2A signaling is a necessary component for the stimulation of ecdysteroidogenesis, potentially by mediating the link between ERK and TOR signaling pathways.

Effects of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family of peptides on MK-801-induced schizophrenia models.

The adipokinetic and red pigment-concentrating hormone (AKH/RPCH) family of peptides controls fat, carbohydrate, and protein metabolism in insects. In our previous study, we showed that AKH possesses antidepressant, anxiolytic, and analgesic effects, causes hyperlocomotion, and exerts neuroprotective effects and increased brain neurotrophic factors in mice. The aim of this study was to investigate the effects of Anax imperator AKH (Ani-AKH), Libellula auripennis AKH (Lia-AKH), and Phormia-Terra hypertrehalosemic hormone (Pht-HrTH) on MK-801-induced memory deterioration in the active allothetic place avoidance test (AAPA) and MK-801-induced sensorimotor gating deficit in the prepulse inhibition test (PPI). In the AAPA task, Long-Evans rats were treated with Ani-AKH (2 mg/kg), Lia-AKH (2 mg/kg), Pht-HrTH (2 mg/kg), MK-801 (0.15 mg/kg), and the combination of MK-801 with the hormones subchronically. In the prepulse inhibition test, Wistar albino rats were treated with Ani-AKH (1 mg/kg), Lia-AKH (1 mg/kg), Pht-HrTH (1 mg/kg), MK-801 (0.1 mg/kg), or the combination of MK-801 with hormones acutely before the test. In our study, Ani-AKH (2 mg/kg), Lia-AKH (2 mg/kg), and Pht-HrTH (2 mg/kg) reversed MK-801 (0.15 mg/kg)-induced cognitive memory impairment effects in the AAPA task. Lia-AKH (1 mg/kg) significantly potentiated the MK-801-induced PPI disruption, while Ani-AKH (1 mg/kg) partially potentiated the impairment caused by MK-801, and Pht-HrTH did not modify the effect of MK-801. In conclusion, AKH had no effect in sensorimotor gating deficits in the PPI test in schizophrenia model while AKH improved memory in the schizophrenia model of MK-801.

GRP78 enabled micelle-based glioma targeted drug delivery.

GRP78, a specific cancer cell-surface marker, is implicated in cancer cells proliferation, apoptosis resistance, metastasis and drug resistance. l-VAP (SNTRVAP) is a tumor homing peptide exhibiting high binding affinity in vitro to GRP78 protein overexpressed on glioma, glioma stem cells, vasculogenic mimicry and neovasculature. Even though short peptides are often non-immunogenic and demonstrate high affinity to tumor cells, their targeting efficacy is always undermined by rapid blood clearance and enzymatic degradation. In the present study, two d peptides RI-VAP (retro inverso isomer of l-VAP) and d-VAP (retro isomer of l-VAP) were developed by structure-guided peptide design and retro-inverso isomerization technique for glioma targeting. RI-VAP and d-VAP were predicted to bind their receptor GRP78 protein with similar binding affinity, which was experimentally confirmed. The results of in vivo imaging demonstrated that RI-VAP and d-VAP had remarkably advantage over l-VAP for tumor accumulation. In addition, RI-VAP and d-VAP modified paclitaxel-loaded polymeric micelle had better anti-tumor efficacy in comparison to taxol, paclitaxel-loaded plain micelles and l-VAP modified micelles. Overall, the VAP modified micelles suggested in the present study could effectively achieve glioma-targeted drug delivery, validating the potential of the stable VAP peptides in improving the therapeutic efficacy of paclitaxel for glioma.

Effects of chronic administration of adipokinetic and hypertrehalosemic hormone on animal behavior, BDNF, and CREB expression in the hippocampus and neurogenesis in mice.

Neurosecretory cells in corpus cardiacum of insects synthesize a set of hormones that are called adipokinetic, hypertrehalosaemic or hyperprolinaemic, depending on insect in question. This study investigated effects of chronic administration of Anax imperator adipokinetic hormone (Ani-AKH), Libellula auripennis adipokinetic hormone (Lia-AKH), and Phormia-Terra hypertrehalosaemic hormone (Pht-HrTH) on depression, anxiety, analgesy, locomotion in forced swimming (FST), elevated plus-maze (EPM), hot plate, and locomotor activity tests. Ani-AKH (1 and 2 mg/kg), Lia-AKH (1 and 2 mg/kg), and Pht-HrTH (1 and 2 mg/kg) had antidepressant effects in forced swimming test. Lia-AKH (2 mg/kg) and Pht-HrTH (1 and 2 mg/kg) had anxiolytic effects when given chronically in elevated plus-maze test. Ani-AKH (1 and 2 mg/kg) and Pht-HrTH (2 mg/kg) had antinociceptive effects in hot plate test in male balb-c mice. Ani-AKH (2 mg/kg), Lia-AKH (1 and 2 mg/kg), and Pht-HrTH had locomotion-enhancing effects in locomotor activity test in male balb-c mice. Drug treatment significantly increased brain-derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) gene expression levels compared to control levels. Pht-HrTH and Ani-AKH groups had significantly increased numbers of BrdU-labeled cells, while neurodegeneration was lower in the Pht-HrTH group. Our study showed that AKH/RPCH family peptides may be used in treatment of psychiatric illness such as depression and anxiety, in treatment of pain and in diseases related to locomotion system. AKH/RPCH family peptides increase neurotrophic factors in brain and have potential proliferative and neuroprotective effects in hippocampal neurogenesis and neurodegeneration.

Bombyx mori prothoracicostatic peptide receptor is allosterically activated via a Gα(i/o)-protein-biased signalling cascade by Drosophila sex peptide.

In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169-31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca²âº mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM-DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM-BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gα(i/o)-dependent cAMP production and away from Ca²âº mobilization and arrestin recruitment.

Isolation and identification of two novel attractant compounds from Chinese cockroach (Eupolyphaga sinensis Walker) by combination of HSCCC, NMR and CD techniques.

High-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hextane-ethyl acetate-methanol-water (1.5:1:1.5:1, v/v/v/v) was applied to the isolation and purification of attractants from Chinese cockroach, Eupolyphaga sinensis Walker. Two new attractants with attractant activity towards the male insects were obtained from the extract sample in a one-step separation. Their purities were determined by HPLC. Subsequent MS, NMR and CD analyses have led to the characterization of (R)-3-ethyl-6,8-dihydroxy-7-methyl-3,4-dihydroisochromen-1-one (1) and (R)-6,8-dihydroxy-3,7-dimethyl-3,4-dihydroisochromen-1-one (2), two novel isocumarin type attractants. Based on these results, it is concluded that HSCCC is a viable separation method option for purifying insect attractants, while effectively maintaining the attracting activity of the isolates. This is the first attempt to apply counter-current chromatography technique to separate attractants from Chinese cockroach.

Neuropeptidergic input pathways to the circadian pacemaker center of the Madeira cockroach analysed with an improved injection technique.

Light entrainment pathways synchronize the circadian clock of almost all species of the animal and plant kingdom to the daily light dark cycle. In the Madeira cockroach Rhyparobia (Leucophaea) maderae, the circadian clock is located in the accessory medulla of the brain's optic lobes. The clock has abundant neuropeptides with unknown functions. Previous studies suggested that myoinhibitory peptides (MIPs), orcokinins (ORCs), and allatotropin (AT) take part in light input pathways to the circadian clock. As the sequences of AT and ORCs of R. maderae have not yet been determined, with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, the respective Rhyparobia peptides were characterized. To search for light-like phase-shifting inputs to the circadian clock, Rhyparobia-MIP-1, Rhyparobia-AT, and Rhyparobia-ORC were injected at different circadian times, combined with locomotor activity assays. An improved, less invasive injection method was developed that allowed for the analysis of peptide effects within <2 weeks after injection. Rhyparobia-MIP-1 and Rhyparobia-AT injections resulted in dose-dependent monophasic phase response curves with maximum delays at the beginning of the subjective night, similar to light-dependent phase delays. In contrast to Manduca sexta-AT, Rhyparobia-AT did not phase advance locomotor activity rhythms. Only injections of Rhyparobia-ORCs resulted in a biphasic light-like phase response curve. Thus, it is hypothesized that Rhyparobia-MIP-1 and -AT are candidates for relaying light-dependent delays and/or non-photic inputs to the clock, whereas Rhyparobia-ORCs might be part of the light-entrainment pathways relaying phase delays and advances to the circadian clock of the Madeira cockroach.

Involvement of phosphorylation of adenosine 5'-monophosphate-activated protein kinase in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.

In this study, we investigated inhibition of the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) by prothoracicotropic hormone (PTTH) in prothoracic glands of the silkworm, Bombyx mori. We found that treatment with PTTH in vitro inhibited AMPK phosphorylation in time- and dose-dependent manners, as seen on Western blots of glandular lysates probed with antibody directed against AMPKα phosphorylated at Thr172. Moreover, in vitro inhibition of AMPK phosphorylation by PTTH was also verified by in vivo experiments: injection of PTTH into day 7 last instar larvae greatly inhibited glandular AMPK phosphorylation. PTTH-inhibited AMPK phosphorylation appeared to be partially reversed by treatment with LY294002, indicating involvement of phosphatidylinositol 3-kinase (PI3K) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside, AICAR) increased both basal and PTTH-inhibited AMPK phosphorylation. Treatment with AICAR also inhibited PTTH-stimulated ecdysteroidogenesis of prothoracic glands. The mechanism underlying inhibition of PTTH-stimulated ecdysteroidogenesis by AICAR was further investigated by determining the phosphorylation of eIF4E-binding protein (4E-BP) and p70 ribosomal protein S6 kinase (S6K), two known downstream signaling targets of the target of rapamycin complex 1 (TORC1). Upon treatment with AICAR, decreases in PTTH-stimulated phosphorylation of 4E-BP and S6K were detected. In addition, treatment with AICAR did not affect PTTH-stimulated extracellular signal-regulated kinase (ERK) phosphorylation, indicating that AMPK phosphorylation is not upstream signaling for ERK phosphorylation. Examination of gene expression levels of AMPKα, ß, and γ by quantitative real-time PCR (qRT-PCR) showed that PTTH did not affect AMPK transcription. From these results, it is assumed that inhibition of AMPK phosphorylation, which lies upstream of PTTH-stimulated TOR signaling, may play a role in PTTH stimulation of ecdysteroidogenesis.

The effects of linear and cyclic analogs of Locmi-DH, Dippu-DH(46) and Dippu-DH(31) on appetitive behavior in Locusta migratoria.

The effects of analogs of the diuretic peptides Locmi-DH, Dippu-DH(46) and Dippu-DH(31) on two aspects of appetitive behavior are investigated in previously food-deprived nymphs of Locusta migratoria. The analogs tested are the C-terminal 15-mer and nonapeptides and their corresponding cyclic analogs. At a nominal dose of 1pmol injected per nymph, the linear fragments and their cyclic analogs of Dippu-DH(46) display no significant effects on the latency to feed or on the length of the first meal in nymphs. However, at the same dose, the linear fragments of Dippu-DH(31) and their cyclic analogs, and analogs of Locmi-DH modulate appetitive behavior: they are anorexigenic in reducing the duration of the first meal, and generally increasing the latency to feed. The cyclic analogs of Dippu-DH(31) are at least as effective as their linear counterparts in influencing these aspects of appetitive behavior in locust nymphs.

Adipokinetic hormone-induced antioxidant response in Spodoptera littoralis.

The antioxidative potential of the Manduca sexta adipokinetic hormone (Manse-AKH) in the last instar larvae of Spodoptera littoralis (Noctuidae, Lepidoptera) was demonstrated after exposure to oxidative stress (OS) elicited by feeding on artificial diet containing tannic acid (TA). Determination of protein carbonyls (PCs) and reduced glutathione (GSH) levels, monitoring of activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione-S-transferases (GSTs), as well as measuring of the mRNA expression of CAT and SOD were used as markers of the OS. Injection of the Manse-AKH (5 pmol per individual) reversed the OS status by mitigation of PCs formation and by stimulation of glutathione-S-transferases (GSTs) activity. The CAT and SOD mRNA expression was significantly suppressed after the Manse-AKH injection while activity of these enzymes was not affected. These results indicate that diminishing of OS after the AKH injection might be a result of activation of specific enzymatic pathway possibly at the post-translational level rather than a direct effect on regulation of antioxidant marker genes at the transcriptional level.

Ecdysis triggering hormone signaling in arthropods.

Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate the ecdysis sequence through action on central neurons expressing ETH receptors (ETHR) in model moth and dipteran species. We used various biochemical, molecular and BLAST search techniques to detect these signaling molecules in representatives of diverse arthropods. Using peptide isolation from tracheal extracts, cDNA cloning or homology searches, we identified ETHs in a variety of hemimetabolous and holometabolous insects. Most insects produce two related ETHs, but only a single active peptide was isolated from the cricket and one peptide is encoded by the eth gene of the honeybee, parasitic wasp and aphid. Immunohistochemical staining with antiserum to Manduca PETH revealed Inka cells on tracheal surface of diverse insects. In spite of conserved ETH sequences, comparison of natural and the ETH-induced ecdysis sequence in the honeybee and beetle revealed considerable species-specific differences in pre-ecdysis and ecdysis behaviors. DNA sequences coding for putative ETHR were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ethr gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). Phylogenetic analysis showed that these receptors fall into a family of closely related GPCRs. We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). The possible source of ETH in ticks was detected in paired cells located in all pedal segments. Our results provide further evidence of structural and functional conservation of ETH-ETHR signaling.

Different actions of ecdysis-triggering hormone on the brain and ventral nerve cord of the hornworm, Manduca sexta.

Ecdysis, or the shedding of the old cuticle, depends on coordinated stereotyped behaviors, regulated by a number of neuropeptides. In the hornworm, Manduca sexta, two neuropeptides interact, namely ecdysis-triggering hormone (ETH) and eclosion hormone. We looked at the effects of ETH in vivo and in vitro, on the brain and the ventral nerve cord to determine the roles played by these hormones. We monitored ecdysis onset and the presence of cGMP and eclosion hormone immunoreactivity. In vivo, only a fraction of larvae lacking the cell bodies containing eclosion hormone, and injected with ETH, were able to undergo ecdysis, with a delayed response. These animals showed strongest cGMP immunoreactivity in the subesophageal and thoracic ganglia, with concomitant reductions in eclosion hormone immunoreactivity in descending axons in comparison with animals not undergoing ecdysis. Animals lacking the brain showed reduced to no cGMP levels in all ganglia. In vitro, isolated CNS preparations lacking the brain initiated ecdysis motor programs after incubation in ETH, with faster onset times than controls, and with reduced cGMP immunoreactivity. If ETH was applied only to the brain of the isolated CNS, cGMP immunoreactivity was noted primarily in the subesophageal and thoracic ganglia, with a decrease in eclosion hormone immunoreactivity in descending axons. ETH addition to the rest of the nerve cord showed reduced eclosion hormone immunoreactivity but little to no cGMP immunoreactivity in any ganglion. Controls showed strong cGMP immunoreactivity in all ganglia, and even greater reductions in eclosion hormone staining after ETH application. These results support previous suggestions that eclosion hormone is required for a positive feedback loop with ETH as well as onset of an inhibitory component, but also suggest that ETH stimulates eclosion hormone release at multiple spike initiation zones. The resultant up regulation of cGMP does not appear to be required for onset of ecdysis. A new model for ecdysis regulation is considered.

Biological activity of diuretic factors on the anterior midgut of the blood-feeding bug, Rhodnius prolixus.

Probing of a host and ingestion of a blood-meal in a fifth instar Rhodnius prolixus results in a cascade of tightly integrated events, including salivary gland secretion, plasticization of the abdominal cuticle, increased ion and water movement across the anterior midgut (crop) and Malpighian tubules (which rapidly produce urine) and the regular expulsion of urine from the hindgut. In this study we have focussed on the role of the anterior midgut during the rapid postprandial diuresis. The huge blood-meal is pumped into the anterior midgut, during feeding, then modified by diuresis and stored until it is digested. Changes in the anterior midgut activity are rapid. Within minutes of the commencement of feeding there is an increase in the frequency of anterior midgut contractions and diuresis begins with the movement of salt and water across the epithelium of the anterior midgut into the haemolymph. While serotonin, a diuretic hormone in R. prolixus, is known to play a role in the physiological activity of the anterior midgut, we were interested in exploring further the role of serotonin, and other diuretic peptides. We have tested the activity of several peptides, including R. prolixus calcitonin-like diuretic hormone (Rhopr-DH 31), corticotropin-releasing factor (CRF)-like peptide from Zootermopsis nevadensis DH (Zoone-DH) and a kinin from Leucophaea maderae, Leucokinin 1 (LK1). These peptides families are known to be present in the central nervous system of R. prolixus, are putative neurohormones released into the haemolymph after the start of feeding, and have been shown to have activity on a variety of tissues involved in post-feeding diuresis. We show here that both serotonin and Zoone-DH increase the cAMP content of the anterior midgut and that serotonin, Zoone-DH and cAMP analogues increase absorption of water from the anterior midgut, increase the short circuit current and voltage, while decreasing the resistance across the epithelium. While LK1 and Rhopr-DH 31 do not significantly increase absorption, or short circuit current, LK1 does significantly decrease the resistance and transepithelial voltage of the anterior midgut epithelium. All of the factors studied increase the frequency of contractions of the anterior midgut.

The synthesis and biological activity of linear and cyclic analogs of the two diuretic peptides of Diploptera punctata.

We have investigated the effect of analogs of the two Dippu diuretic hormones, Dippu-DH(46) and Dippu-DH(31), on fluid secretion by Malpighian tubules of male Diploptera punctata. We synthesized analogs containing the amino acid methyl-homoserine, to replace methionine residues, to render these modified peptides less subject to oxidation. We have also synthesized C-terminal fragments and their corresponding cyclic analogs to determine their effect on fluid secretion in D. punctata. Our results indicate that the modified peptides retain significant activity in the Ramsay secretion assay. The linear fragments displayed no activity or some inhibitory activity whereas the cyclic analog fragments showed stimulatory activity, in the case of DH(46), or slight inhibitory activity, in the case of DH(31).

Endocrinology of reproduction and phase transition in locusts.

In the last decade, important progress has been made in the experimental analysis of the endocrine mechanisms controlling reproduction and phase transition in locusts. Phase transition is a very fascinating, but complex, phenomenon of phenotypic plasticity that is triggered by changes in population density and can lead to the formation of extremely devastating hopper bands and adult gregarious locust swarms. While some phase characteristics change within hours, others appear more gradually in the next stage(s), or even in the next generation(s). In adults, the phase status also has a major influence on the process of reproduction. A better understanding of how solitarious locusts become gregarious and how this switch affects reproductive physiology may result in novel strategies to fight locust plagues. In this paper, we will review the current knowledge concerning this close interaction between locust phase polyphenism and reproduction.

Expression of lipid storage droplet protein-1 may define the role of AKH as a lipid mobilizing hormone in Manduca sexta.

Adipokinetic hormone (AKH) is the main hormone involved in the acute regulation of hemolymph lipid levels in several insects. In adult Manduca sexta AKH promotes a rapid phosphorylation of "Lipid storage protein-1", Lsd1, and a concomitant activation of the rate of hydrolysis of triglycerides by the main fat body lipase. In contrast, in the larval stage AKH modulates hemolymph trehalose levels. The present study describes the sequence of a full-length Lsd1 cDNA obtained from M. sexta fat body and investigates a possible link between Lsd1 expression and the distinct effects of AKH in larva and adult insects. The deduced protein sequence showed a high degree of conservation compared to other insect Lsd1s, particularly in the central region of the protein (amino acids 211-276) in which the predicted lipid binding helices are found. Lsd1 was absent in feeding larva and its abundance progressively increased as the insect develops from the non-feeding larva to adult. Contrasting with the levels of protein, Lsd1 transcripts were maximal during the feeding larval stages. The subcellular distribution of Lsd1 showed that the protein exclusively localizes in the lipid droplets. Lsd1 was found in the fat body but it was undetectable in lipid droplets isolated from oocytes or embryos. The present study suggests a link between AKH-stimulated lipolysis in the fat body and the expression of Lsd1.

Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH31) from Rhodnius prolixus.

Diuresis in the blood-gorging hemipteran Rhodnius prolixus is under neurohormonal control and involves a variety of processes and tissues. These include ion and water movement across the epithelium of the crop and the Malpighian tubules, and muscle contractions of the crop, hindgut and dorsal vessel, which facilitate mixing of the blood-meal, mixing of the haemolymph, as well as the expulsion of waste. One of the neurohormones that might play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH(31)) family of insect peptides. Previously we have demonstrated the presence of DH(31)-like peptides in the central nervous system (CNS) and gut of R. prolixus 5th instars. In the present work, a DH(31) from the CNS of 5th instar R. prolixus was isolated using reversed-phase liquid chromatography (RPLC), monitored with an enzyme-linked immunosorbent assay (ELISA) combined with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, and sequenced using tandem mass spectrometry and Edman degradation. This neuropeptide is the first to be sequenced in R. prolixus and has a sequence identical to that found previously for Dippu-DH(31) from the cockroach Diploptera punctata. In previous studies testing Rhopr/Dippu-DH(31) in Malpighian tubule secretion assays, we demonstrated increases in the rate of secretion that were small, relative to that induced by serotonin, but nevertheless 14-fold over baseline. In the present study, we investigated second messenger pathways in response to Rhopr/Dippu-DH(31) and found no increase or decrease in cyclic adenosine monophosphate (cyclic AMP) content of the Malpighian tubules. DH(31)-like immunoreactivity is present over the dorsal hindgut, anterior dorsal vessel and dorsal diaphragm, and bioassays of the R. prolixus dorsal vessel and hindgut indicate that Rhopr/Dippu-DH(31) increases the frequency of muscle contractions of both tissues. Second messenger pathways were also investigated for the dorsal vessel and hindgut.

Octopamine partially restores walking in hypokinetic cockroaches stung by the parasitoid wasp Ampulex compressa.

When stung by the parasitoid wasp Ampulex compressa, cockroaches Periplaneta americana enter a hypokinetic state that is characterized by little, if any, spontaneous locomotor activity. In the present study we investigate the effect of an octopamine receptor agonist and an antagonist on the locomotor behavior of stung and control cockroaches. We show that in cockroaches stung by a wasp the octopamine receptor agonist chlordimeform induces a significant increase in spontaneous walking. In good agreement, in control individuals an octopamine receptor antagonist significantly reduces walking activity. Adipokinetic hormone I (AKH-I) promotes spontaneous walking in controls but does not do so in stung individuals, which suggests that the venom effect is most probably not mediated by AKH-I. Dopamine receptor agonists or antagonists had no significant effect on the spontaneous walking of stung or control cockroaches, respectively. The effect of the octopamine receptor agonist was maximal when injected into the brain, suggesting that the wasp venom interferes with octopaminergic modulation of walking initiation in central structures of the cockroach brain.