Crustacean molt-inhibiting hormone: structure, function, and cellular mode of action.

In Crustacea, secretion of ecdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks. This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a) structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis. Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.

Molecular characterization and cell-specific expression of an ion transport peptide in the tobacco hornworm, Manduca sexta.

The crustacean hyperglycemic hormone (CHH) peptides regulate diverse physiological processes from reproduction to metabolism and molting in arthropods. In insects, the ion transport peptides (ITP), also members of the CHH family, have only been implicated in ion transport. In this study, we sequenced a nucleotide fragment spanning the conserved A1/A2 region of the putative CHH/ITP gene. This fragment was amplified from larval cDNA of the tobacco hornworm, Manduca sexta and showed a high degree of sequence conservation with the same region from other insects and, to a lesser degree, with that of crustacean species, suggesting the presence of a Manduca-specific CHH/ITP mRNA (MasITP mRNA). CHH-like immunocytochemical analyses with two crustacean antisera (from Carcinus maenas and Cancer pagurus) identified the presence of CHH-like immunoreactivity in nervous tissue of all developmental stages, but not in the gut of M. sexta. Specifically, CHH-like peptides localized to paired type IA(2) neurosecretory cells of the pars lateralis of the brain (projecting ipsilaterallly to the corpora cardiaca-allata complex) and to neurosecretory cells and transverse nerves of the ventral nerve cord in larvae, pupae, and adults. The distribution of the putative MasITP peptide shifted during development in a manner consistent with metamorphic reorganization. A comparison of hemolymph equivalents of CHH detected by enzyme-linked immunosorbent assay with CHH-like immunoreactivity in transverse nerves provided evidence for the release of MasITP from the transverse nerves into the hemolymph at insect ecdysis. These data suggest the presence of an insect ITP in M. sexta and a role for this hormone during ecdysis.

Is crustacean hyperglycaemic hormone precursor-related peptide a circulating neurohormone in crabs?

Sites of synthesis and release patterns of crustacean hyperglycaemic hormone precursor-related peptide (CPRP) were investigated with those of crustacean hyperglycaemic hormone (cHH), in order to determine whether this precursor-related peptide satisfies certain criteria necessary for its definition as a secretable, circulating hormone. Using the edible crab, Cancer pagurus, sites of CPRP synthesis were determined by immunohistochemistry and release patterns of both peptides were determined in vivo and in vitro by radioimmunoassay of haemolymph and eyestalk superfusates. Both peptides were co-released from sinus glands (SGs) following potassium-evoked depolarization of isolated eyestalk preparations. However, stress-evoked in vivo release resulted in apparent non-stoichiometric circulating peptide profiles. This phenomenon is explained by notable differences in clearance rates of the peptides in haemolymph. In contrast to cHH, CPRP is very slowly degraded in vivo. Although CPRP is clearly a circulating peptide, whose release is concomitant with that of cHH, physiologically pertinent roles for this molecule remain to be discovered.

Expression of the crustacean hyperglycaemic hormones and the gonad-inhibiting hormone during the reproductive cycle of the female American lobster Homarus americanus.

Crustacean reproduction is regulated by a complex chain of hormonal interactions in which the crustacean hyperglycaemic hormones A and B (CHH-A and CHH-B) and the gonad-inhibiting hormone (GIH) play a primary role. These neurohormones are produced in the same neuroendocrine cells of the X-organ sinus gland complex, situated in the eyestalks of the American lobster, Homarus americanus. In order to obtain more information on the synthesis, storage, release and function of these three neuropeptides during the reproductive cycle, we studied the levels of their mRNAs in the X-organ, their peptide storage in the sinus gland and their concentration in the haemolymph at different stages of the female reproductive cycle. A high CHH-A mRNA level was found only in the previtellogenic stage, while elevated mRNA levels were determined for CHH-B in the mature as well as the previtellogenic stage. High CHH storage levels in the sinus gland were found during previtellogenesis. The total amount of CHH (CHH-A plus -B) in the haemolymph was significantly higher during maturation. A low level of GIH mRNA in the X-organ and a low amount of the GIH I isoform in the sinus gland were found only in the immature stage. In contrast, GIH haemolymph levels were high during the immature and previtellogenic stages. We conclude that CHH-A and -B are involved in triggering the onset of vitellogenesis and that CHH-B in particular is responsible for stimulating oocyte maturation before spawning, while GIH prevents the start of vitellogenesis in the ovary. Moreover, our results show that the balance between the haemolymph levels of the CHHs and GIH may tune the synchronization of reproduction and molting during the biannual reproductive cycle of the American lobster.

Comparison of early and late treatment with a recombinant endotoxin neutralizing protein in a rat model of Escherichia coli sepsis.

OBJECTIVE: To test the efficacy of a recombinant endotoxin neutralizing protein as compared with saline in rats with Escherichia coli sepsis. DESIGN: Prospective, controlled animal trial. SETTING: Hospital animal research laboratory. SUBJECTS: Male Wistar rats challenged with intraperitoneal E. coli, O18ac K1, and treated 1 hr later with ceftriaxone and gentamicin. INTERVENTIONS: Recombinant endotoxin neutralizing protein, 50 mg/kg, was administered to rats 1, 2, or 3 hrs after E. coli challenge; saline was administered to control animals. MEASUREMENTS AND MAIN RESULTS: Quantitative bacteremia, 1 hr after challenge and before antibiotic administration, was not significantly different between treatment groups (range geometric mean 451 to 621 colony-forming units [cfu]/mL). The endotoxin concentration, measured immediately before recombinant endotoxin neutralizing protein administration, was significantly higher in animals sampled and treated at 2 hrs (geometric mean 260 EU/mL; 95% confidence interval 140 to 480 EU/mL), or 3 hrs (geometric mean 697 EU/mL; 95% confidence interval 307 to 1585 EU/mL) after E. coli challenge, compared with animals sampled and treated at 1 hr (geometric mean 17 EU/mL; 95% confidence interval 7 to 69 EU/ mL). Survival rate was significantly greater in rats treated with recombinant endotoxin neutralizing protein at 1 hr (23/27; p < .001) or 2 hrs (8/30; p < .01) after E. coli challenge than in controls (1/32). CONCLUSION: Administration of recombinant endotoxin neutralizing protein delayed up to 2 hrs after challenge with E. coli improves survival in antibiotic-treated rats with Gram-negative sepsis.

Sequestration of insecticyanin, a blue hemolymph protein, into the egg of the hawkmoth Manduca sexta. Evidence for receptor-mediated endocytosis.

Sequestration of the blue biliprotein, insecticyanin, into developing oocytes of the hawkmoth, Manduca sexta was investigated. Immunodiffusion assays revealed that insecticyanin concentration in mature eggs (29.6 microM) is slightly higher than that in hemolymph (25.8 microM). The endocytotic uptake of insecticyanin was visualized at the light microscopic level using autoradiography. Uptake of 125I-insecticyanin by isolated oocytes was saturable. Analysis of in vitro uptake data estimated that the value of K(uptake) (insecticyanin concentration at half-maximal uptake rate) is 4.2 microM and that the Vmax (maximum rate of uptake) is 1 pmol follicle-1 h-1. Labeled insecticyanin was shown to bind to sonicated follicle membranes with high specificity and affinity. The KD (equilibrium dissociation constant) and the Bm (total number of binding sites per follicle), were estimated as 4 x 10(-8) M and 8 x 10(7) respectively. Competition studies showed that binding of labeled insecticyanin to oocyte membranes was blocked by excess amounts of unlabeled insecticyanin but not by lipophorin and vitellogenin of M. sexta. Additional membrane binding experiments demonstrated that receptors for insecticyanin are only present in the oocytes membranes, not in fat body or gut tissue.

Isolation, characterization and developmental expression of the ecdysteroid-induced E75 gene of the wax moth Galleria mellonella.

Using the cDNA for the Drosophila ecdysteroid-induced member of the steroid-hormone-receptor superfamily, E75A, we isolated a genomic clone from Galleria mellonella that revealed 77% similarity with the region of E75A cDNA encoding the C-terminal zinc-finger motif. A Galleria cDNA clone was isolated that encoded a complete DNA-binding domain composed of two zinc fingers and designated GmE75A. Its deduced amino acid sequence showed 100% and 85% identities within the DNA-binding and ligand-binding domains of Drosophila E75A, respectively. The Galleria genomic clone did not encode the N-terminal zinc finger, but included a sequence similar to the B1 exon, which is unique to the B isoform of E75. Thus, the cDNA and genomic DNA sequences indicated that the Galleria gene E75 encoded at least two isoforms, GmE75A and GmE75B, which differed in their N-termini. Probes specific for GmE75A and B hybridized to two distinct transcripts of 2.6 kb. Both GmE75A and B mRNA levels correlated closely with the ecdysteroid titer during development. At the onset of larval/pupal transformation, both transcripts appeared in high amounts within 4 h of the ecdysteroid rise, then declined concurrently with the hormone titer decline. At the time of pupal ecdysis, there was another peak of GmE75A expression but not GmE75B expression, coincident with a minor ecdysteroid pulse. In isolated abdomens of final instar larvae, GmE75A mRNA was induced by 20-hydroxyecdysone within 20 min of the injection; the mRNA levels were maximal at 1 h and declined by 3 h following the treatment.

Effects of the avermectin analogue MK-243 on vitellogenesis and reproduction in the ixodid tick, Amblyomma hebraeum.

A dose-dependent reduction in weight of total egg mass and a slightly increased oviposition latency were observed following injection of the avermectin analogue MK-243 (4"-epi-methylamino-4"-deoxyavermectin B1) directly into the haemocoel of engorged female Amblyomma hebraeum Koch (Acari: Ixodidae). Egg laying was almost completely inhibited at 100 micrograms/kg body weight. MK-243 markedly inhibited ovary development and vitellogenesis. Ticks treated with 100 micrograms MK-243/kg also had one-tenth the haemolymph ecdysteroid concentration compared to controls 10 days post-engorgement. Thus, among its other effects on ticks, the avermectins also inhibit the process of vitellogenesis.

Decapitation or starvation raise haemolymph ecdysteroid titres in the ovoviviparous female cockroach, Blaberus craniifer Burm.

1. Decapitating newly emerged Blaberus craniifer females near the prothorax severs connections between the suboesophageal and prothoracic ganglia, thus depriving them of the neuroendocrine cephalic complex (including brain and suboesophageal ganglion) and the anterior end of prothoracic glands (PGs). 2. As demonstrated by enzyme immunoassay (EIA), headless females have higher levels of ecdysteroids (ECDs) in haemolymph than starved or fed females, indicating that the neuroendocrine cephalic complex influences circulating ECD levels. 3. The time course of hormonal peaks in decapitated females resembles that in starved females during the first post-ecdysial week, suggesting that some as yet unknown regulating mechanism of ECD production lies outside the head. 4. It is suggested that: (a) The PGs are sites for ECDs production in the early post-imaginal period, (b) the prothoracic and suboesophageal ganglia (linked by nerves to PGs) regulate PGs activity, possibly via neural inputs.

Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistasin and tick anticoagulant peptide (TAP) in a rabbit model of venous thrombosis.

An in vivo thromboplastin (TP)-induced venous stasis thrombosis model in rabbits was used to compare the efficacy of standard heparin with the selective factor Xa inhibitors, recombinant tick anticoagulant peptide (rTAP) and recombinant antistasin (rATS), in prophylactic prevention of thrombus formation. Heparin significantly reduced TP-induced clot formation at doses of 55 and 100 U kg-1h-1 yielding clot weights of 9 +/- 4 and 6 +/- 2%, respectively. Clot formation was significantly decreased by i.v. infusions of rTAP at doses of 21, 37 and 64 micrograms kg-1 min-1 resulting in normalized clot weights of 13 +/- 3, 8 +/- 2 and 2 +/- 1%, respectively. rATS was approximately 10-fold more potent than rTAP, reducing normalized clot weights to 16 +/- 5, 2 +/- 1 and 1 +/- 0.8% at rATS doses of 1.25, 2.5 and 5.0 micrograms kg-1 min-1, respectively. These data suggest that factor Xa-mediated inhibition of coagulation with rTAP and rATS is as effective as conventional anticoagulant treatment with heparin in preventing venous thrombosis.

Investigation of ecdysteroid excretion by adult Dirofilaria immitis and Brugia pahangi.

The excretion of ecdysteroids by the filarial nematode species, Dirofilaria immitis and Brugia pahangi, was examined both in vitro, by the analysis of culture medium, and in vivo, through analysis of serum samples from experimentally infected hosts. There was no evidence of ecdysteroid excretion by intact parasites of either species in vitro. Free ecdysteroids were detected in the serum of ferrets and dogs infected with D. immitis, but concentrations would be at or below the limit of detection in sub-millilitre serum samples. The detection of ecdysteroids in the serum of potential hosts is unlikely to be of value in the diagnosis of filarial infections due to a combination of low titre in the presence of current infection and measurable titre in its absence. Ecdysteroids of dietary origin may contribute to the latter.

Hypogonadism and ecdysteroid production in Loa loa and Mansonella perstans filariasis.

Possible endocrinological repercussions of infection with Loa loa and Mansonella perstans filariae were studied in Gabonese subjects. Microfilaremic males were compared with amicrofilaremic controls. In the infected group 13/105 subjects (12%) presented only abnormally low serum levels of testosterone (less than 4 ng/ml), 25/105 (24%) only abnormally high serum levels of gonadotrophins, FSH (greater than 15 mIU/ml) and LH (greater than 20 mIU/ml), and 22/105 (21%) presented anomalies in both testosterone and gonadotrophin levels. One out of 68 control subjects had 3.6 ng/ml seric testosterone and all had normal levels of gonadotrophins. Ecdysteroids were detected (greater than 0.025 ng/ml) in the serum of 87/97 (90%) microfilaremic subjects (GM 0.123 ng/ml) compared to 12/64 (19%) controls (GM 0.030 ng/ml). Ecdysteroids were detected in the urine of all subjects, infected (GM 8.468 ng/ml) as well as control (GM 1.245 ng/ml). The hormonal perturbations were correlated with the levels of Loa loa microfilaremia but not with those of serum and urinary ecdysteroids. These results demonstrate that microfilaremic subjects often show endocrinal signs of hypogonadism and present appreciable levels of ecdysteroids in serum and urine. A direct role for parasitic ecdysteroids in hypogonadism remains to be demonstrated.

Purification of a beta-1,3-glucan recognition protein in the prophenoloxidase activating system from hemolymph of the silkworm, Bombyx mori.

The plasma fraction (referred to as plasma-CPB) of silkworm hemolymph, from which a protein with affinity to beta-1,3-glucan was specifically removed according to Yoshida et al. (Yoshida, H., Ochiai, M., and Ashida, M. (1986), Biochem. Biophys. Res. Commun. 141, 1177-1184), was used to develop a method for quantitating the beta-1,3-glucan recognition protein of the prophenoloxidase activating system. In principle, a sample was judged to contain beta-1,3-glucan recognition protein when that sample could restore the ability of the system in plasma-CPB to be triggered by beta-1,3-glucan. Purification procedures for the recognition protein from silkworm hemolymph consisted of fractionation with ammonium sulfate, chromatography on DEAE-Toyopearl, Affi-Gel-heparin, and Mono Q and Superose 12 on the fast protein liquid chromatography system of Pharmacia LKB Biotechnology Inc. About 2.03 mg of beta-1,3-glucan recognition protein was obtained from 300 ml of hemolymph. The purified beta-1,3-glucan recognition protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing-polyacrylamide gel electrophoresis. beta-1,3-Glucan recognition protein had a molecular mass of 62 kDa composed of a single polypeptide and an isoelectric point of pH 4.3. It bound to curdlan beads (composed of beta-1,3-glucan with average particle size of 80 micron) in the absence of divalent cation, whereas its binding to glucans with beta(1----4)- or alpha(1----6)-glycosidic linkages was not detected under the experimental conditions. Elution of the beta-1,3-glucan recognition protein bound to curdlan beads could be achieved under strongly denaturing conditions (after incubation of the beads with sodium dodecyl sulfate and beta-mercaptoethanol in boiling water for 5 min), but elution at room temperature was poor. Since beta-1,3-glucan recognition protein is the only protein in silkworm plasma with strong affinity to beta-1,3-glucan and endows the prophenoloxidase activating system in plasma-CPB with the ability to be triggered by beta-1,3-glucan, it was concluded that binding of the purified beta-1,3-glucan recognition protein with beta-1,3-glucan causes the triggering of the prophenol-oxidase activating system in silkworm plasma. However, the nature of the activity that is generated as the result of binding is not yet known. The purified beta-1,3-glucan recognition protein bound to beta-1,3-glucan did not hydrolyze appreciably any of the 26 commercially available peptidyl-7-amino-4-methylcoumarins, substrates for various proteases.

Demonstration of a proallatotoxin-sensitive period in 4th-instar nymphs of Rhodnius prolixus.

1. Topical application of the proallatotoxin ethoxyprecocene II (60 micrograms/nymph) to 4th-instar nymphs of Rhodnius prolixus at various times before and after a blood meal induced precocious metamorphosis. 2. The highest percentage of adultoids was obtained when proallatotoxin was applied prior to feeding or up to day 3 after the blood meal. 3. The proallatotoxin-sensitive period ended 4 days after feeding just before the maximum peak of ecdysteroid concentration in the hemolymph. 4. The significance of these findings is discussed in terms of the juvenile hormone-sensitive period and the hormonal program which controls molting in this insect.

Regulation of the stress-responsive X-organ--Y-organ axis by 5-hydroxytryptamine in the crab, Cancer antennarius.

Eyestalk-intact, but not de-eyestalked, crabs (Cancer antennarius) given five injections of 5-hydroxytryptamine (5HT, 750 micrograms per injection) in 48 hr showed significant reductions in hemolymph ecdysteroid titers within the first 12 hr which continued through Hour 72; the effect was reversible (Hours 72-144). The 5HT synthesis inhibitor p-chlorophenylalanine (PCPA; 60 micrograms) or the 5HT receptor antagonist cyproheptadine (CPH; 100 micrograms) caused significant rises in serum ecdysteroids in intact, but not in de-eyestalked, crabs; with four injections over 48 hr, a rise was evident at 12 hr, continued through 72 hr, and returned to control levels within 4 days post-treatment. Stress due to handling depressed ecdysteroid levels, an effect that was reversed by PCPA and CPH; values in the treated groups returned to prestress levels within 24 hr and were significantly greater than prestress levels within 48 hr. 5HT treatment did not further decrease stress-reduced ecdysteroid titers. Normal behaviors are described in posturing and in defensive responses to stress; these were changed in both intact and de-eyestalked crabs by 5HT treatment (but not by PCPA or CPH). These findings support the hypothesis that release of molt-inhibiting hormone (MIH) from crab eyestalks is mediated by 5HT and suggest that stress induces 5HT-mediated MIH release.

Physiological approach to the onset of receptivity in female Acheta domesticus. I. Role of the corpora allata and ovaries.

During the 32 hr following the imaginal moult, all female Acheta domesticus actively or passively refuse male courtship; they are unreceptive. As of 32 hr, the most precocious females become receptive and accept mating. At this time, juvenile hormone (JH III) synthesized by corpora allata (CA) is already detectable in hemolymph, while ecdysteroids (synthesized by ovaries) begin increasing at 48 hr. JH III and ecdysteroid levels in hemolymph were measured by RIA. After allatectomy and/or ovariectomy, all females became receptive, thus showing that CA and/or ovaries are not essential to the onset of receptivity. However, male courtship is longer for allatectomized females; in ovariectomized females, mating is delayed.

Normal and experimentally induced changes in hormonal hemolymph titers during parental behavior of the earwig Labidura riparia.

The reproductive cycle of Labidura riparia includes two distinct phases of behavior: a feeding and sexual phase followed by a parental and fasting phase. These phases correspond to two contrasting physiological phases (vitellogenesis, followed by ovarian inactivity). These correlations have been verified by correlating radioimmunoassay (RIA) measurements of the levels of circulating juvenile hormones (JH) and ecdysteroids with ovarian state during the first reproductive cycle. Similar studies were also made after experimentally suppressing parental activity (care of eggs) either by depriving females of their eggs or by force-feeding during the egg-care phase. Taking eggs away without feeding caused the disappearance of parental behavior and a short lived period of vitellogenesis. Likewise, feeding in the presence of eggs resulted in vitellogenesis and disappearance of egg-care behavior. Thus, it appears that in order for the parental phase to develop normally it is necessary to preserve the proper external conditions (the presence of eggs) and physiological conditions (fasting).

Changes in ecdysteroid and juvenile hormone titers in the hemolymph of Galleria mellonella larvae and pupae.

The variations in circulating ecdysteroids and juvenile hormones (JH) in Galleria, from the end of the antepenultimate larval stage until emergence of adults, have been determined. The two hormonal families were extracted separately from the same hemolymph sample and quantified by two radioimmunoassays. Juvenile hormone RIA activity was about 35 nM in larvae of the antepenultimate and penultimate stages. It dropped before each molt and increased thereafter. Moreover, it gradually decreased during the last larval instar. In pupae, it was generally low, but it rose drastically during the late pupal development and in young adults. This rise was very much higher in females than in males. Three different RIA-active compounds were found; they were assumed to be JH-I, JH-II, and JH-III according to their retention times in HPLC. The three compounds were present in almost equal concentration in larvae of the penultimate stage: JH-I predominated, however, during the last larval instar. In late pupae, the main hormone was JH-III both in males and in females. There is no clear relationship between ecdysteroid and juvenile hormone changes, except for a female-specific ecdysteroid rise which coincides with the juvenile hormone release in late pupae. This double hormonal stimulation can be involved in the regulation of vitellogenin synthesis and deposition in oocytes.