Release Of Crustacean Hyperglycemic Hormone Research Articles

Impacts of ZnO and multi-walled carbon nanotubes (MWCNTs) on biological parameters of the Oriental river prawn Macrobrachium nipponense De Haan, 1849 (Decapoda: Caridea: Palaemonidae)

Abstract Nanomaterials (NMs) mainly settle on bottom sediments and accumulate in the benthos of aquatic communities. Decapod crustaceans, being deposit-omnivorous feeders, are appropriate biological models to assay the biological effects of NMs. The present study evaluated the effects of ZnO and multi-walled carbon nanotubes (MWCNTs) NMs separately and in combination on the activities of antioxidant enzymes on crustacean hyperglycemic hormone (CHH), hematology, reproductive performance, and hepatopancreas lesions the Oriental river prawn, Macrobrachium nipponense De Haan, 1849 [De Haan, 1833–1850]. We essayed concentrations of ZnO (0, 1, 10, 30, 50 mg l–1) and MWCNTs (0, 5, 10, 15, 20 mg l–1) and two higher concentrations of the NMs were compared in combined treatments. Both NMs showed dose-dependent effects on reproductive parameters of M. nipponense, especially the fertilization rate of eggs. Inter-molt and inter-spawn periods were postponed and reproduction stopped in 50 ppm ZnO and 15 and 20 ppm MWCNTs treatments. Our findings showed that the release of crustacean hyperglycemic hormone (CHH) and superoxide dismutase (SOD) enzyme activity are directly related to the NMs levels independently as well as in combination. NMs did not show any effect on the activities of alanine transaminase (ALT) and aspartate transaminase (AST) enzymes in muscle tissue independently, but the combined effects of the activities of these enzymes were significant. NMs caused hepatocyte enlargement, melanization, apoptosis, and necrosis damages in the hepatopancreas of prawns. The findings showed that ZnO and MWCNTs NMs have strong adverse biological effects on M. nipponense, and that this species is an appropriate bio-indicator and bio-monitor organism for NMs contaminations in the freshwater aquatic environments.

Inhibiting viral replication and prolonging survival of hosts by attenuating stress responses to viral infection

Viruses are obligate intracellular parasites relying on host cells to obtain biosynthetic precursors and energy to successfully infect the host. The metabolic profile of the host cell is known to be altered in response to viral infection to satisfy the resources demanded during viral replication. Previous data of ours showed that white spot syndrome virus (WSSV) elicited in a crustacean host (Procambarus clarkii) a rapid and long-lasting release of crustacean hyperglycemic hormone (CHH), a well-known carbohydrate-regulating and stress response-mediating endocrine hormone. Therefore, the WSSV-enhanced release of CHH could be responsible at least in part for the metabolic alterations in the WSSV-challenged host. To investigate the possible metabolic roles of CHH in the host-parasite interaction, we studied whether silencing CHH gene expression could inhibit WSSV propagation in tissues and reduce the mortality of the WSSV-infected animals. Data presented in this study showed that CHH gene silencing indeed resists the WSSV infection. Injection of CHH dsRNA at the dosage of 140 μg/g BW caused significant decreases of viral copy number in tissues of WSSV-infected host, particularly showing a pronounced effect in the endodermal tissues (including hepatopancreas and gastrolith disk). Furthermore, results from the cumulative mortality showed that the treatment of CHH dsRNA delayed death from WSSV. Injection of CHH dsRNA at the dosages of 70, 17, and 10 μg/ g BW significantly extended the mean survival time. Together, this study concludes that the silencing of the CHH gene does have an inhibitory effect on the replication of the white spot syndrome virus and can assist the host to mitigate the invasion of WSSV, through attenuating CHH-mediated stress responses.

Effects of ZnO nanoparticles on the Giant freshwater prawn (Macrobrachium rosenbergii, de Man, 1879): Reproductive performance, larvae development, CHH concentrations and anti-oxidative enzymes activity

The Giant freshwater prawn (Macrobrachium rosenbergii) breeds when in captive conditions. The eggs of a clutch are attached to the abdomen of berried females. Zinc oxide (ZnO) is one of the most important metal oxide-nanoparticles (NPs) that is widely used in various industries and is released into aquatic environments from wastewater management facilities. The present study was conducted to evaluate effects of ZnO on values for the reproductive variables: larvae development, crustacean hyperglycemic hormone (CHH) release from the X-organ into the hemolymph and anti-oxidative enzymes activity of M. rosenbergii. There were five groups including a group not treated (control), and groups treated with10, 20, 50, 100 mg/L ZnO in triplicate during a 90-day period. Results indicated that ZnO-NPs have marked effects on reproductive performance, offspring development, CHH release from the X-organ into the hemolymph and anti-oxidant enzymes activities with there being no spawning of brood-stock in the 100 mg/L ZnO group and in the prawns treated with 50 mg/L there was spawning but there was larvae mortality immediately subsequent to hatching. Also, values for viability rate of eggs, dry weight of eggs, brood-stock inter-spawn period and egg clutch somatic index (ESI) reproductive variables were affected by the NP. This NP was found to have a dose-dependent effect on CHH release from the X-organ into the hemolymph and also superoxide dismutase (SOD) and catalase activities in M. rosenbergii. The results indicate that M. rosenbergii, a freshwater decapod crustacean, is an appropriate species to study nano-material effects on reproduction in freshwater ecosystems.

Hyperglycemic and osmotic effects of dopamine and recombinant hormone CHH-B1 in the Pacific white shrimp Litopenaeus vannamei

The effects of dopamine (DA) on crustacean hyperglycemic hormone (CHH) release and osmoregulation were investigated in the white shrimp Litopenaeus vannamei. Application of 2 μg of the recombinant CHH-B1His hormone or 2 × 10−6 mol L−1 of DA to intact shrimp caused an increase in the hemolymph glucose levels 1 h post-injection, suggesting that DA might stimulate hyperglycemia through CHH release from the sinus glands. This assumption was supported by similar experiments using bilaterally eyestalk-ablated shrimp. Additionally, rCHH-B1His restored the osmoregulatory capacity (OC) of shrimp under hyperosmotic conditions to basal values 2 h post-injection, whereas DA led to an OC decrease in shrimp at all sampling times. These neuroendocrine factors may be involved in the control of metabolism and osmoregulation in L. vannamei and could be important for its adaptation to different environmental conditions.

Crustacean hyperglycemic hormones of two cold water crab species, Chionoecetes opilio and C. japonicus: Isolation of cDNA sequences and localization of CHH neuropeptide in eyestalk ganglia

Crustacean hyperglycemic hormone (CHH) is primarily known for its prototypical function in hyperglycemia which is induced by the release of CHH. The CHH release takes place as an adaptive response to the energy demands of the animals experiencing stressful environmental, physiological or behavioral conditions. Although >63 decapod CHH nucleotide sequences are known (GenBank), the majority of them is garnered from the species inhabiting shallow and warm water. In order to understand the adaptive role of CHH in Chionoecetes opilio and Chionoecetes japonicus inhabiting deep water environments, we first aimed for the isolation of the full-length cDNA sequence of CHH from the eyestalk ganglia of C. opilio (ChoCHH) and C. japonicus (ChjCHH) using degenerate PCR and 5′ and 3′ RACE. Cho- and ChjCHH cDNA sequences are identical in 5′ UTR and ORF with 100% sequence identity of the putative 138aa of preproCHHs. The length of 3′ UTR ChjCHH cDNA sequence is 39 nucleotides shorter than that of ChoCHH. This is the first report in decapod crustaceans that two different species have the identical sequence of CHH. ChoCHH expression increases during embryogenesis of C. opilio and is significantly higher in adult males and females. C. japonicus males have slightly higher ChjCHH expression than C. opilio males, but no statistical difference. In both species, the immunostaining intensity of CHH is stronger in the sinus gland than that of X-organ cells. Future studies will enable us to gain better understanding of the comparative metabolic physiology and endocrinology of cold, deep water species of Chionoecetes spp.

Qualitative and quantitative top‐down mass spectral analysis of crustacean hyperglycemic hormones in response to feeding

An efficient pipeline for peptide discovery accelerates peptidomic analysis and facilitates a better understanding of the functional roles of neuropeptides. However, qualitative and quantitative analysis of large neuropeptides is challenging due to the bigger molecular sizes, multiple PTMs, and interference by homologous isoforms. Herein, we refined two methodologies in the pipeline for highly confident and efficient MS-based peptide discovery. For the qualitative analysis, the so-called "high resolution/accurate mass" measurement on Orbitrap mass spectrometers was integrated with computer-assisted homology search, which was successfully applied to decipher the substituted amino acid residues in large neuropeptides by referring to homologous sequences. For the quantitative analysis, a new isotopic labeling-assisted top-down MS strategy was developed, which enabled direct monitoring of the abundance changes of endogenous large neuropeptides. By using the refined peptide discovery pipeline, one novel crustacean hyperglycemic hormone (CHH) from the Dungeness crab sinus glands was confidently identified and de novo sequenced, and its relative abundance was quantified. Comparative analysis of CHHs in unfed and fed crabs revealed that the peptide abundance in the sinus glands was significantly increased after food intake, suggesting that the release of CHHs might be altered by feeding behavior.

An insulin-like growth factor found in hepatopancreas implicates carbohydrate metabolism of the blue crab Callinectes sapidus

Hyperglycemia that is caused by the release of crustacean hyperglycemic hormone (CHH) from the sinus gland to hemolymph is one of the hallmark physiological phenomena, occurring in decapod crustaceans experiencing stressful conditions. However, the mechanism(s) by which such elevated glucose levels return to resting levels is still unknown. Interestingly, noted is a difference in the clearance rate of hemolymph glucose between adult females and adult males of the blue crab, Callinectes sapidus: the former with more rapid clearance than the latter. The presence of an endogenous-insulin-like molecule is suggested in C. sapidus because an injection of bovine insulin, significantly reduces the levels of hemolymph glucose that were previously elevated by emersion stress or the glucose injection. Using 5′ and 3′ RACE, the full-length cDNA of an insulin-like molecule is isolated from the hepatopancreas of an adult female C. sapidus and shows the same putative sequence of an insulin-like androgenic gland factor (IAG) but differs in 5′ and 3′ UTR sequences. A knock-down study using five injections of double-stranded RNA of CasIAG-hep (dsRNA-CasIAG-hep, 10μg/injection) over a 10-day period reduces CasIAG-hep expression by ∼50%. The levels of hemolymph glucose are also kept higher in dsRNA-CasIAG-hep injected group than those treated with dsRNA-green fluorescent protein (dsRNA-IAG-hep) or saline. Most importantly, the hepatopancreas of dsRNA-CasIAG-hep injected animals contains amounts of carbohydrate (glucose, trehalose, and glycogen) significantly lower than those of control groups, indicating that the function of CasIAG-hep in carbohydrate metabolism in crustaceans is similar to carbohydrate metabolism in vertebrates.

Neuroendocrine responses of a crustacean host to viral infection: Effects of infection of white spot syndrome virus on the expression and release of crustacean hyperglycemic hormone in the crayfish Procambarus clarkii

The objectives of the present study were to characterize the changes in crustacean hyperglycemic hormone (CHH) transcript and peptide levels in response to infection of white spot syndrome virus (WSSV) in a crustacean, Procambarus clarkii. After viral challenge, significant increase in virus load began at 24h post injection (hpi) and the increase was much more substantial at 48 and 72hpi. The hemolymph CHH levels rapidly increased after viral challenge; the increase started as early as 3hpi and lasted for at least 2d after the challenge. In contrast, the hemolymph glucose levels did not significantly changed over a 2 d period in the WSSV-infected animals. The CHH transcript and peptide levels in tissues were also determined. The CHH transcript levels in the eyestalk ganglia (the major site of CHH synthesis) of the virus-infected animals did not significantly change over a 2 d period and those in 2 extra-eyestalk tissues (the thoracic ganglia and cerebral ganglia) significantly increased at 24 and 48hpi. The CHH peptide levels in the eyestalk ganglia of the virus-infected animals significantly decreased at 24 and 48hpi and those in the thoracic ganglia and cerebral ganglia remained unchanged over a 2 d period. These data demonstrated a WSSV-induced increase in the release of CHH into hemolymph that is rapid in onset and lasting in duration. Changes in the CHH transcript and peptide levels implied that the WSSV-induced increase in hemolymph CHH levels primarily resulted from an enhanced release from the eyestalk ganglia, but the contribution of the 2 extra-eyestalk tissues to hemolymph pool of CHH increased as viral infection progressed. The combined patterns of change in the hemolymph glucose and CHH levels further suggest that the virus-enhanced CHH release would lead to higher glycolytic activity and elevated glucose mobilization presumably favorable for viral replication.

Serotonin modulation of hemolymph glucose and crustacean hyperglycemic hormone titers in Fenneropenaeus indicus

Crustacean hyperglycemic hormone (CHH), a neurohormone synthesized and released from the X-organ sinus gland complex, is primarily involved in carbohydrate metabolism. Biogenic amines and peptidergic neuroregulators are known to modulate the release of CHH. Although mechanisms involved in hormone release in the shrimp, Fenneropenaeus indicus is poorly understood. Daily variations in the levels of crustacean hyperglycemic hormone and serotonin in the shrimp F. indicus were studied. Both CHH and glucose levels increase during the first hours after the beginning of darkness. The animals were injected with serotonin and glycemic and CHH levels were determined after 2 h of injection. Serotonin caused significant hyperglycemia ( P < 0.0.5) in both intact and eyestalk ablated shrimp. These results support the hypothesis that serotonin produces hyperglycemia in crustaceans by triggering release of hyperglycemic hormone both from the eyestalk and from the other sites of its release.

Dynamics of in Vivo Release of Molt-Inhibiting Hormone and Crustacean Hyperglycemic Hormone in the Shore Crab, Carcinus maenas

Very little is known regarding the release patterns or circulating titers of neuropeptides in crustaceans, in particular those concerned with regulation of molting hormone (ecdysteroid) synthesis, molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH), which is also an adaptive hormone, centrally important in carbohydrate metabolism. Furthermore, the currently accepted model of molt control is founded on an untested hypothesis suggesting that molting can proceed only after decline in MIH titer. Accordingly, we measured simultaneous circulating neuropeptide profiles for both MIH and CHH by RIA of purified hemolymph during the molt cycle at fine temporal scale during day/night cycles and seasonally. For CHH we additionally determined release patterns after physiologically relevant stress. Results show that both hormones are released exclusively and episodically, rather than continuously, with notably short half-lives in circulation, suggesting dynamic and short-lived variations in levels of both hormones. During the molt cycle, there are no overt changes in MIH titer, except a massive and unprecedented increase in MIH during late premolt, just before ecdysis. The function of this hormone surge is unknown. Treatment with various stressors (hypoxia, temperature shock) showed that CHH release occurs extremely rapidly, within minutes of stress. Release of CHH after stressful episodes during premolt (when gut endocrine cells synthesize large quantities of CHH) is exclusively from the sinus gland: CHH from the gut is never involved in the stress response. The results show a hitherto unsuspected dynamism in release of MIH and CHH and suggest that currently accepted models of molt control must be reconsidered.

Enkephalinergic control of the secretory activity of neurons producing stereoisomers of crustacean hyperglycemic hormone in the eyestalk of the crayfish Orconectes limosus.

A subgroup of neurons in the classical X organ sinus gland neuroendocrine system of the crayfish (Orconectes limosus) eyestalk produces two chiral forms of the crustacean hyperglycemic hormone (CHH) in two different types of neurons: CHH in 22 cells and D Phe(3) CHH in eight cells. Previous reports have demonstrated that release of CHH from the sinus gland is inhibited by enkephalins. Here, we have addressed the questions of 1) whether this inhibition affects one or both types of CHH neurons, 2) where the site of enkephalinergic control of CHH and/or D Phe(3) CHH is, and 3) whether the inhibitory effect is due to direct or indirect interactions of enkephalinergic neurons with CHH cells. In vitro incubations of neurosecretory complexes followed by immunoassays of CHH isoforms indicated that both methionine and leucine enkephalins inhibit release of the two CHH isoforms from crayfish eyestalks, by a receptor mediated process. Whole mount double or triple immunofluorescence labelings combined with confocal microscopy revealed enkephalin immunostaining in all neuropils of the eyestalk, except in the sinus gland. Virtual thin confocal sections showed many close appositions between terminals of enkephalinergic neurons and dendritic arborizations of specific CHH immunoreactive cells in the medulla terminalis neuropil. This provides the first evidence for direct inputs from enkephalinergic neurons into dendrites of both CHH cell types, which suggests that enkephalins inhibit release of both CHH isoforms via synaptic contacts.

Serotonergic regulation of crustacean hyperglycemic hormone secretion in the crayfish, Procambarus clarkii.

These studies investigate if crustacean hyperglycemic hormone (CHH) is involved in 5-hydroxytryptamine (5-HT)-induced hyperglycemia. Eyestalk ganglia with intact X-organ-sinus gland complex were dissected from the crayfish Procambarus clarkii and incubated under various experimental conditions. Incubation media were then analyzed for the presence of released hyperglycemic factor using an in vivo bioassay. The results show that 5-HT enhanced release of hyperglycemic factor in a dose-dependent manner. This stimulatory effect of 5-HT was significantly decreased by adding ketanserin or methysergide (both 5-HT receptor antagonists) into incubation of eyestalk ganglia. Further, activity of the 5-HT-released hyperglycemic factor could be eliminated by adsorption of incubation media with anti-CHH serum but not by preimmune or anti-5-HT serum. These results confirm the hypothesis that 5-HT enhances release of CHH, which in turn elicits hyperglycemic responses. It is probable that 5-HT activates an excitation-secretion coupling mechanism by interacting with receptors located on the X-organ neurosecretory cells.

Carbohydrate Dynamics and the Crustacean Hyperglycemic Hormone (CHH): Effects of Parasitic Infection in Norway Lobsters (Nephrops norvegicus)

The effects of a dinoflagellate parasite (Hematodinium sp.) on carbohydrate metabolism were examined in the Norway lobster, Nephrops norvegicus. Five stages of infection were observed. These included uninfected (Stage 0), subpatently infected (SP), and patently infected (Stage 1–4) lobsters. During patent infection, the concentration of glucose in the hemolymph was reduced significantly from its value of 180 μg ml−1 in uninfected (Stage 0) lobsters to 25.3 μg ml−1 in Stage 3–4. These changes were accompanied by significantly lower levels of hepatopancreatic glycogen in lobsters at Stage 2 (2.01 mg g−1) and Stage 3–4 (0.84 mg g−1) of infection than in those at Stage 0 (16.19 mg g−1) and Stage 1 (14.71 mg g−1). Due to disruption of the normal feedback loops which control the release of crustacean hyperglycemic hormone (CHH), plasma concentrations increased with the severity of infection from 32.2 fmol ml−1 in Stage 0 to 106.6 fmol ml−1 in Stage 3–4. The increased CHH concentrations occurred concomitantly with reduced concentrations of plasma glucose and tissue glycogen. A significantly increased hemolymph CHH titer (107.7 fmol ml−1) was also observed during SP infection. It is concluded that the parasite places a heavy metabolic load on the host lobster.

Hyperglycemic responses to cold shock in the freshwater giant prawn, Macrobrachium rosenbergii

Crustacean hyperglycemic hormone (CHH), a neurohormone synthesized and released from the x-organ sinus gland complex, is primarily involved in carbohydrate metabolism; biogenic amines and peptidergic neuroregulators are known to modulate the release of CHH. Marked elevations of hemolymph glucose titers, which peaked within 2 h, were observed in both intact and bilaterally eyestalk-ablated prawns, Macrobrachium rosenbergii, when they were transferred directly from their optimal temperature of 28 °C to lower temperatures close to their lethal limit. Hyperglycemia can therefore be considered a characteristic response in this species under cold shock. Involvement of biogenic amines in the hyperglycemic response was also demonstrated. Hyperglycemic effects of epinephrine, dopamine and serotonin were mediated through CHH at the eyestalk level, but the response under cold shock was not exclusively mediated through CHH. It is suggested that factor(s) other than CHH are involved in the hyperglycemic response, possibly norepinephrine or/and octopamine.

Variation in Larval Size After Eyestalk Ablation in Larvae of the Snapping Shrimp Alpheus Heterochaelis

The effect of eyestalk ablation at various times on size (overall body length, carapace length, and body width) of fourth instar (postlarval instar during normal development) Alpheus heterochaelis was studied. Animals which underwent bilateral eyestalk ablation earlier in larval life grew to a significantly larger overall size compared to unablated controls and larvae ablated later. This growth was manifested as lengthening of the abdomen and was not expressed in the cephalothorax. The relationship between total body length attained and time of ablation was greatest among larvae ablated during Stage II, with overall size decreasing as time of ablation increased chronologically. The increase in overall size seen among larvae ablated progressively earlier may be a result of loss of hormonal control over hydromineral regulation and/or controlled manufacture and release of crustacean hyperglycemic hormone (CHH) by the X-organ-sinus gland, but it may also simply reflect a redirection of metabolic resources.

D-glucose-sensitive neurosecretory cells of the crab Cancer borealis and negative feedback regulation of blood glucose level.

We studied the effects of glucose on cultured X-organ neurons of the crab Cancer borealis using single-electrode current- and voltage-clamp techniques. A subpopulation of the cells responded to D-glucose with a hyperpolarization. These cells, but not glucose-insensitive cells, showed immunoreactivity to crustacean hyperglycemic hormone (CHH), the hormone responsible for the elevation of blood glucose levels in crustaceans. Glucose-sensitive cells were also inhibited by serotonin and gamma-aminobutyric acid but were not affected by dopamine and Leu-enkephalin. The response was specific for D-glucose, with an EC50 of 0.25 mmoll-1. No response was seen to L-glucose, sucrose, galactose, mannose or fructose. The glucose response persisted in the absence of extracellular Na+ and in low-Ca2+/Mn2+ saline. In voltage-clamp experiments, D-glucose evoked a small current with a reversal potential close to that of voltage-dependent K+ currents. We conclude that D-glucose activates a K+ current in CHH-immunoreactive cells that, in normal saline, induces a hyperpolarization. We propose that this enables glucose to regulate directly the release of CHH into the hemolymph, thus constituting a negative feedback mechanism regulating hemolymph glucose concentration.

Dopaminergic and Enkephalinergic Involvement in the Regulation of Blood Glucose in the Red Swamp Crayfish, Procambarus clarkii

In in vivo experiments injection of dopamine (DA) or leucine enkephalin (L-Enk) produced hypoglycemia in intact red swamp crayfish, Procambarus clarkii . In contrast neither compound had a significant effect on the circulating glucose concentration in eyestalkless individuals. With intact crayfish spiperone, a DA receptor blocker, inhibited this hypoglycemic action of DA but not this action of L-Enk. In contrast, in intact crayfish naloxone, a competitive opioid antagonist, inhibited the in vivo hypoglycemic actions of both L-Enk and DA. In vitro, both DA and L-Enk reduced the rate of release of the crustacean hyperglycemic hormone (CHH) front isolated eyestalk neuroendocrine tissue. These results suggest that both DA and L-Enk act to inhibit release of CHH by affecting the eyestalk neuroendocrine complex and that the enkephalinergic inhibitory neuron follows the dopaminergic inhibitory neuron in the chain of neurons which leads to the neuroendocrine cells that secrete CHH.

NEUROSECRETION OF CRUSTACEAN HYPERGLYCEMIC HORMONE EVOKED BY AXONAL STIMULATION OR ELEVATION OF SALINE K+ CONCENTRATION QUANTIFIED BY A SENSITIVE IMMUNOASSAY METHOD

A sandwich-type enzyme-linked immunosorbent assay (ELISA) was utilized to quantify crustacean hyperglycemic hormone (as Carcinus maenas equivalents) released by single X-organ&shy;sinus gland systems of the crab Cardisoma carnifex during continuous perifusion. Basal rates of secretion (20&shy;60 pg min-1) were stable for at least 4 h. Electrical stimulation (600 stimuli in 5 min) of the axon tract increased secretion two- to threefold, but only if it resulted in neural activity that was propagated to the terminals of the sinus gland. No difference was observable when stimuli were given repetitively or as a series of trains. Perifusion with saline having ten times the normal K+ concentration augmented secretion by as much as fivefold. Augmented secretion of crustacean hyperglycemic hormone evoked by either electrical or K+ stimulation appeared abruptly but declined slowly (over tens of minutes) after stimulation was stopped. K+-evoked secretion of crustacean hyperglycemic hormone was maintained without decrement for at least 1 h. Basal secretion increased in saline from which Ca2+ had been omitted, but decreased in saline containing Mn2+. Neither electrical stimulation nor high [K+] augmented secretion in Ca2+-deficient saline or if Mn2+ was present. Introduction of Mn2+ during K+-evoked secretion immediately reduced release to unstimulated levels; secretion resumed promptly upon removal of Mn2+. Tetrodotoxin reversibly blocked both electrical and secretory responses to axonal stimulation, but it did not block basal or K+-evoked secretion. Release of crustacean hyperglycemic hormone by isolated axon terminals was augmented two- to threefold by perifusion with saline having ten times the normal K+ concentration. The responses were similar to those of the intact systems, having a rapid onset, well-maintained secretion and a long 'tail' of secretion after removal of the K+ stimulus.

The role of biogenic amines in the control of blood glucose level in the decapod crustacean, Carcinus maenas L.

1. Administration of biogenic amines into intact Carcinus maenas induces dose- and timedependent elevation of hemolymph glucose level. 2. Removal of the neurosecretory centre containing the crustacean hyperglycemic hormone (CHH) by ablation of the eyestalks did not induce hypoglycemia. 3. Injection of dopamine (DA) into eyestalkless crabs showed no hyperglycemic effect, while serotonin (5-HT), epinephrine (E), norepinephrine (NE), and octopamine (OA) elevated glucose levels. 4. The dopaminergic effect was significantly reduced by administration of trifluoperazine (TFP). 5. 5-HT and OA were found to be strong elevators of glucose levels, while the other biogenic amines had moderate effects only. 6. The results indicate, that DA exerts its hyperglycemic effect by stimulating the release of CHH from the eyestalk neurosecretory centre. Elevation of hemolymph glucose level by 5-HT, OA, E, and NE, occurs independently of CHH.

Isolation, sequence analysis, and physiological properties of enkephalins in the nervous tissue of the shore crab Carcinus maenas L.

[Leu]- and [Met]enkephalin from thoracic ganglia of the shore crab Carcinus maenas have been purified to homogeneity by a reversed-phase HPLC procedure. Automated gas-phase sequencing revealed a primary structure identical with that of enkephalins from vertebrates. The immunoreactive material in different parts of the nervous system of C. maenas was measured by RIA. Isolated crustacean [Leu]enkephalin shows physiological properties identical to synthetic [Leu]enkephalin in our bioassay. The enkephalinergic effect has been monitored by two bioassays: (i) inhibition of release of crustacean hyperglycemic hormone from isolated eyestalks of C. maenas, and (ii) decreased effect on blood glucose level in the fiddler crab Uca pugilator. The decrease in blood sugar level induced by [Leu]enkephalin was antagonized by naloxone, indicating a receptor-mediated process. In sequence analysis, the thoracic ganglion yielded 3 pmol of [Leu]enkephalin and approximately 1 pmol of [Met]enkephalin. The presence of enkephalins in crustaceans shows an earlier phylogenetic origin of opioids than presumed.