Levels Of Crustacean Hyperglycemic Hormone Research Articles

The impact of chasing stress on the metabolism of the Atlantic Ghost Crab Ocypode quadrata (Fabricius, 1787).

Ocypode quadrata, a Ghost crab species found along the western Atlantic coast, is considered a bioindicator of anthropogenic impact on sandy beaches. Ghost Crabbing, a touristic activity in which ghost crabs are chased just for fun, is a potentially threatening activity for this crab. In crustaceans, metabolites such as glucose and lactate, and the gene expression of crustacean hyperglycemic hormone (CHH) and heat shock proteins (HSPs) increase when the animals are exposed to several types of stress, including alterations in temperature, salinity, or exposure to xenobiotics. This work was developed to identify if being chased by humans would affect these markers of stress in this species of crab. The effects of chasing stress on hemolymph and tissue metabolites and the gene expression levels of CHH and HSP70 were investigated. The levels of lactate in the hemolymph of stressed crabs were six times higher than those of control crabs immediately after chasing and decreased progressively during recovery, indicating an active anaerobic metabolism during the stress. On the contrary, glucose levels in the hemolymph of the stressed crabs increased progressively from 30 to 60 min after chasing, indicating an inverse correlation between glucose and lactate and the conversion of lactate to glucose by gluconeogenesis. In stressed crabs, the levels of triglycerides in the hemolymph decreased 30 min after chasing, while the opposite tended to occur in the hepatopancreas, indicating that during recovery, the crabs use triglycerides as energy source to sustain aerobic metabolism. Finally, this study demonstrates that ghost crabs are stressed by minimum human contact and that "ghost crabbing" must not be encouraged as a tourist activity.

Experimental effects of cadmium on physiological response of Callinectes danae (Crustacea, Portunidae) from environments with different levels of Cd contamination

Cadmium (Cd) can adversely affect aquatic life, altering reproductive and molting processes in crustaceans. The objective of this study was to evaluate the influence of Cd on reproduction and molting in the crab Callinectes danae. Adult females were obtained from environments with different levels of pollution: low (LC), medium (MC), and high contaminated (HC) areas. Animals from LC, MC, and HC areas were exposed to 0, 0.5, and 2 mg L−1 of CdCl2 for 3 h. Cd bioaccumulation, oxidative stress (evaluated by antioxidant enzymes activity), and lipid peroxidation (LPX) were analyzed in mature ovaries (stage II), gills, and hepatopancreas. The expression levels of crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) genes were quantified in the eyestalks, while 17β-estradiol (E2) and melatonin concentration were measured in the hemolymph. Cd bioaccumulated mainly in the hepatopancreas and gills, with increased E2, LPX, and antioxidant enzymes in HC compared to the LC region. Decreased CHH and MIH transcripts were observed in the animals from HC regions compared to LC and MC areas. Physiological differences were recorded, especially for bioaccumulation, oxidative stress, and hormone levels, in animals sampled in HC areas compared to LC and MC regions. In conclusion, the physiological damage triggered by Cd could be reduced due to higher levels of melatonin and antioxidant enzymes in HC areas.

The exploration of neuroendocrine regulation of crustacean hyperglycemic hormone (CHH) on innate immunity of Litopenaeus vannamei under ammonia-N stress

To unveil the neuroendocrine-immune (NEI) mechanism of crustaceans under high ambient ammonia-N, crustacean hyperglycemic hormone (CHH) in L. vannamei was knocked down under 20 mg/L ammonia-N exposure. The results showed that the expression of CHH in the eyestalks decreased significantly when CHH was silenced. After CHH was knocked down, the levels of CHH, ACh, DA, NE, and 5-HT in the haemolymph decreased significantly. Correspondingly, the expressions of GC, ACh7R, DM1, DA1R, and 5-HT7R in haemocytes down-regulated significantly, while DA4R and α2AR up-regulated significantly. Besides, the expression of Toll3 reduced significantly. And significantly changes occurred in the levels of G protein effectors (AC and PLC), second messengers (cAMP, cGMP, CaM, and DAG), protein kinases (PKA, PKC and PKG), and nuclear transcription factors (CREB, Dorsal, Relish and NKRF). Furthermore, immune defense proteins (BGBP and PPO3, Crustin A, ALF, LYC, TNFα, and IL-16), phagocytosis-related proteins (Cubilin, Integrin, Peroxinectin, Mas-like protein, and Dynamin-1) and exocytosis-related proteins (SNAP-25, VAMP-2 and Syntaxin) changed significantly. Eventually, a significant decrease in the levels of THC, haemocytes phagocytosis rate, plasma PO, antibacterial and bacteriolytic activities was detected. Therefore, these results indicate that under ammonia-N stress, the combination of CHH and GC mainly affects exocytosis of shrimp through the cGMP-PKG-CREB pathway. Simultaneously, CHH stimulates the release of biogenic amines, and then activate G protein effectors after binding to their specific receptors, to regulate exocytosis mainly via the cAMP-PKA-CREB pathway and influence phagocytosis primarily by the cAMP-PKA-NF-κB pathway. CHH can enhance ACh, and then activate G protein effectors after binding to the receptors, and finally regulate exocytosis mainly through the cAMP-PKA-CREB pathway and regulate phagocytosis by the cAMP-PKA-NF-κB pathway. CHH can also promote Toll3-NF-κB pathway, thereby affecting the expressions of immune defense factors. This study contributes to a further understanding of the NEI mechanism of crustacean in response to environmental stress.

Thermal stress induced hyperglycemia in the blue swimmer crab, Portunus pelagicus

Hyperglycemia is a stress responsible mechanism induced in crustaceans through the secretion of Crustacean Hyperglycemic Hormone (CHH). The effect of thermal shock on the hemolymph CHH levels was studied in P. pelagicus. Crabs were exposed to varying temperatures for 3h and were then transferred to ambient temperature (28°C). A higher CHH level of 47.30±2.26fmol/ml was observed on exposure of crabs to 24°C, over a recovery period of 3h. This was reflected with increase in hemolymph glucose causing hyperglycemia and subsequent decrease in hepatopancreas glycogen levels. The results suggest the modulatory role of CHH in producing the energy required for the physiological reparation faced by the crabs during thermal stress.

Gamma-aminobutyric acid regulates glucose homeostasis and enhances the hepatopancreas health of juvenile Chinese mitten crab (Eriocheir sinensis) under fasting stress

The ability of immune defense and resistance to physiological stress is crucial to animal health and survival. This study investigated the regulation of γ-aminobutyric acid (GABA) on metabolic homeostasis and its enhancement of hepatopancreas health in juvenile Chinese mitten crab (Eriocheir sinensis) under food deprivation. Juvenile crabs of 400 individuals were divided into four treatment groups: a control group without injection, and injections with a phosphate-buffered saline solution, 100 μmol GABA/mL and 1000 μmol GABA/mL, respectively. Hypoglycemia was induced by fasting, whereas the GABA treatment regulated hemolymph glucose homeostasis. The quantitative real-time PCR (qRT-PCR) results showed that the GABA treatment significantly up-regulated the mRNA expression levels of crustacean hyperglycemic hormone (CHH) and pyruvate kinase (PK). In contrast, the expression of E. sinensis insulin-like peptide (EsILP) was significantly down-regulated in the cranial ganglia, thoracic ganglia and hepatopancreas. Moreover, acid phosphatase (ACP), alkaline phosphatase (AKP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were significantly increased in the hepatopancreas by the GABA treatment. Furthermore, the hemocyanin content in serum was significantly increased with the GABA injection, and the glutathione (GSH) content, total superoxide dismutase (T-SOD) activity and catalase (CAT) activity in the hepatopancreas showed a similar increasing trend with the dose elevation of GABA. Therefore, these results indicate that GABA can effectively maintain the hemolymph glucose homeostasis by regulating the levels of glucose metabolism-related hormones and key enzymes to promote the degradation and utilization of hepatopancreas glycogen. Meanwhile, GABA can improve the hepatopancreas function and immune status of juvenile E. sinensis under fasting stress. The treatment with GABA may provide a clue to guide health management in crab farming.

Molecular cloning of crustacean hyperglycemic hormone (CHH) family members (CHH, molt-inhibiting hormone and mandibular organ-inhibiting hormone) and their expression levels in the Jonah crab, Cancer borealis

The crustacean hyperglycemic hormone (CHH) neuropeptide family has multiple functions in the regulation of hemolymph glucose levels, molting, ion, and water balance and reproduction. In crab species, three neuroendocrine tissues: the eyestalk ganglia (medulla terminalis X-organ and -sinus gland = ES), the pericardial organ (PO), and guts synthesize a tissue-specific isoforms of CHH neuropeptides. Recently the presence of the mandibular organ-inhibiting hormone (MOIH) was reported in the stomatogastric nervous system (STNS) that regulates the rhythmic muscle movements in esophagus, cardiac sac, gastric and pyloric ports of the foregut. In this study, we aimed to determine the presence of a tissue-specific CHH isoform in the Jonah crab, Cancer borealis using PCR with degenerate primers and 5′, 3′ rapid amplification of cDNA ends (RACE) in the ES. PO, and STNS. The analysis of CHH sequences shows that C. borealis has one type of CHH isoform, unlike other crab species. We also isolated the cDNA sequence of molt-inhibiting hormone (MIH) in the ES and MOIH in the ES and STNS. The presence of CHH, MOIH and MIH in the sinus gland of adult females and males is confirmed by using a dot-blot assay with the putative peaks collected from RP-HPLC and anti-Cancer sera for CHH, MIH, and MOIH. The present of crustacean female sex hormone (CFSH) in the sinus gland of adult females was examined with a dot-blot assay with anti-Callinectes CFSH serum. Levels of CHH, MOIH, and MIH in the sinus gland and their expressions in the eyestalk ganglia are estimated in the adult males, where CHH is the predominant form among these neuropeptides.

Crustacean hyperglycemic hormone (CHH) regulates the ammonia excretion and metabolism in white shrimp, Litopenaeus vannamei under ammonia-N stress

To understand the adaptation of Litopenaeus vannamei to high environmental ammonia-N, RNA interference was used to investigate the function of crustacean hyperglycemic hormone (CHH) in the physiological process of neuroendocrine signaling transduction, and ammonia excretion and metabolism. The shrimp were exposed to 25 mg/L NH4Cl and injected with 20 μg/shrimp CHH dsRNA for 72 h. The results showed that hemolymph ammonia content increased under ammonia-N stress and further increased after CHH knockdown, suggesting that CHH can promote ammonia excretion. Moreover, after CHH knockdown, the levels of CHH, DA, and Wnts decreased significantly, the expression of receptor GC, DA1R, Frizzled and LRP 5/6 also decreased, while DA4R increased remarkably. Moreover, PKA and PKG decreased, while PKC markedly increased, and nuclear transcription factors (CREB and TCF) as well as effector proteins (β-catenin, FXYD2, and 14–3-3) were significantly downregulated. Furthermore, ammonia transporters Na+/K+-ATPase (NKA), K+channel, Rh protein, AQP, V-ATPase, and VAMP decreased significantly, while Na+/H+ exchangers (NHE) and Na+/K+/2Cl− cotransporter (NKCC) increased significantly. These results suggest that CHH regulates ammonia excretion in three ways: 1) by mainly regulating ion channels via PKA, PKC, and PKG signaling pathways; 2) by activating related proteins primarily through Wnt signaling pathway; and 3) by exocytosis, mostly induced by the PKA signaling pathway. In addition, the levels of Gln, uric acid, and urea increased in accordance with the activities of GDH/GS, XDH, and arginase, respectively, suggesting that ammonia excretion was inhibited but ammonia metabolism was slightly enhanced. This study deepens our understanding of the mechanism by which crustaceans respond to high environmental ammonia-N.

Heavy metal stress induced hyperglycemia in blue swimmer crab, Portunus pelagicus

The study was to find out the effect of cadmium and mercury on levels of hemolymph glucose, crustacean hyperglycemic hormone (CHH) and hepatopancreas glycogen in the blue swimmer crab Portunus pelagicus. The experiments were performed in both intact and eyestalk ablated crabs. Quantification of CHH was done by the indirect ELISA with the aid of primary anti-Carcinus maenas-CHH antibody. Higher glucose concentration was observed on exposure to 8×10–6 of cadmium ((825.6±5.42) μg/mL) and 6×10–6 of mercury ((90.5±6.25) μg/mL) after 48 h and 24 h respectively. Higher level of hemolymph glucose was observed in eyestalk intact crabs on exposure to cadmium and mercury than eyestalk ablated crabs. Decrease in the levels of CHH was observed in both eyestalk intact and ablated crabs on heavy metal exposure. Decline of the hepatopancreas glycogen level was also witnessed with the exposure to heavy metal, which validated its utilization in the production of glucose. Thus this study brings to light, the variations in hemolymph glucose, CHH and hepatopancreas glycogen on heavy metal stress. These carbohydrate metabolites can be used as biomarkers in assessing heavy metal contamination in water bodies.

Metabolic effects of parasitization by the barnacle Polyascus plana (Cirripedia: Rhizocephala: Sacculinidae) on a grapsid host, Metopograpsus thukuhar.

Pathophysiological studies of rhizocephalan infections are rare. We describe differences in the levels of tissue and hemolymph metabolites between Polyascus plana-parasitized and unparasitized individuals of Metopograpsus thukuhar. Crabs were assigned to either a parasitized (carrying at least 1 externa, i.e. a protruding reproductive body) or an unparasitized (not carrying externae and determined to be rootlet-free by a barnacle 18S rRNA-based polymerase chain reaction) group. Quantification of metabolites showed that muscle glycogen levels were significantly lower and hepatopancreas levels were significantly higher in parasitized crabs compared to unparasitized crabs; hepatopancreas triacylglycerol levels were significantly higher and hemolymph levels significantly lower in parasitized hosts, and there was no significant difference in muscle triacylglycerol levels between unparasitized and parasitized animals. Glucose levels in the hepatopancreas, muscle, and hemolymph were all significantly higher in parasitized hosts. Significant levels of glucose, triacylglycerol, and glycogen were present in the barnacle externae. In addition, levels of crustacean hyperglycemic hormone in the sinus glands were not significantly different between unparasitized and parasitized animals. Glucose mobilized from the muscle is likely converted to glycogen and triacylglycerol in the rootlet-infiltrated hepatopancreas of parasitized hosts, and the eyestalk neuroendocrine system appears not to be significantly impaired, in terms of hormone production and storage, by parasitization.

Effects of starvation on energy metabolism and crustacean hyperglycemic hormone (CHH) of the Atlantic ghost crab Ocypode quadrata (Fabricius, 1787)

Ocypode quadrata, a crab species found in sandy beaches along the western Atlantic coast, spends the winter months underground without feeding. The main objective of this work was to determine whether O. quadrata is naturally adapted to food deprivation for a period of time. The effects of starvation on the energy metabolism and the expression levels of crustacean hyperglycemic hormone (CHH), known to have an adaptive role in response to many types of environmental stresses, were investigated. First, we isolated the full-length cDNA sequence of OcqCHH and localized the cells producing CHH neuropeptide in eyestalk ganglia by immunostaining. Second, the levels of OcqCHH transcripts were determined in the fed and starved (15 days) intermolt crabs using qRT-PCR assay. The concentration of carbohydrate and lipids in the hemolymph, muscle and hepatopancreas was measured. OcqCHH cDNA sequence has the typical structure of CHHs, and its expression did not change by starvation. Starvation decreased hemolymphatic glucose, muscular glycogen and hepatopancreatic lipids. This implies that hepatopancreas lipolysis followed by β-oxidation, hepatopancreas gluconeogenesis and muscular glycogenolysis may be responsible for the energy requirements of O. quadrata during nutritional stress. Taken together, these results suggest that O. quadrata may be adapted to nutritional deprivation for an extended period of time.

Ecdysteroids regulate the levels of Molt-Inhibiting Hormone (MIH) expression in the blue crab, Callinectes sapidus.

Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR) in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml) and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w) ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle.

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.

Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement

To understand the hormonal coordination of the antagonism between molting and reproduction in crustaceans, the terminally anecdysial mature female Callinectes sapidus was used as a model. The regulatory roles of crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) in vitellogenesis were examined. A competitive specific RIA was used to measure the levels of MIH and CHH in the hemolymphs of mature females at pre- and mid- vitellogenic stages, and their effects on vitellogenesis at early (early 2, E2) and mid vitellogenesis (3) stages were determined in vitro. A hepatopancreas fragments incubation system was developed and the levels of vitellogenin (VtG), as well as VtG mRNA and heterogeneous nuclear (hn)VtG RNA were determined using RIA or QPCR, respectively. MIH titers were four times higher at mid-vitellogenesis than at pre-vitellogenesis, while CHH levels in the hemolymph were constant. In the in vitro incubation experiments, MIH increased both VtG mRNA levels and secretion at ovarian stage 3. At stage E2, however, MIH resulted in a mixed response: downregulation of VtG mRNA and upregulation of hnVtG RNA. CHH had no effect on any of the parameters. Actinomycin D blocked the stimulatory effects of MIH in stage 3 animals on VtG mRNA and VtG, while cycloheximide attenuated only VtG levels, confirming the MIH stimulatory effect at this stage. MIH is a key endocrine regulator in the coordination of molting and reproduction in the mature female C. sapidus, which simultaneously inhibits molt and stimulates vitellogenesis.

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.

Effect of serotonin on hemolymph glucose regulation in the fresh water edible crab Oziotelphusa senex senex

One of the well known crustacean hormones is the crustacean hyperglycemic hormone (CHH). However, the mechanisms involved in the release of this hormone into circulation are poorly studied. The fresh water crabs Oziotelphusa senex senex were injected with serotonin and hemolymph glucose and CHH levels were determined. Injection of serotonin into intact crabs caused significant hyperglycemia in a dose-dependent manner. Administration of serotonin also caused a significant increase in the circulatory levels of CHH. In contrast, injection of serotonin did not cause any change in hemolymph glucose level in eyestalk ablated crabs. The results confirm that serotonin induced hyperglycemia was apparently mediated by the eyestalk hormone CHH.

Binding sites of crustacean hyperglycemic hormone and its second messengers on gills and hindgut of the green shore crab, Carcinus maenas: A possible osmoregulatory role

To determine the possible involvement of crustacean hyperglycemic hormone (CHH) in osmoregulation in crustaceans, ligand binding and second messenger assays were performed on gills and hindgut preparations of the green shore crab Carcinus maenas, whilst midgut gland, previously known as one of the target tissues of CHH served as a control tissue. Classical receptor binding analyses using [ 125I]CHH by saturation and displacement experiments from membrane preparations from gills, hindgut, and midgut glands demonstrated that CHH binding characteristics involved one site, highly specific, saturable, and displaceable kinetics: (gills: K D 5.87 ± 2.05 × 10 −10 and B MAX 6.50 ± 1.15 × 10 −10, hindgut: K D 3.54 ± 1.49 × 10 −10 and B MAX 2.31 ± 0.44 × 10 −10, and midgut gland: K D 7.28 ± 0.9 × 10 −10 and B MAX 3.28 ± 0.25 × 10 −10) all expressed as M/mg protein. No differences, in terms of displacement were observed between the two CHH isoforms (N-terminally blocked pGlu and unblocked Gln) variants. CHH binding sites appeared to be coupled to a second messenger system involving cGMP in all the tissues examined. Exposure of crabs to dilute seawater increased levels of cGMP, glucose in gills and circulating CHH levels. Other crustacean neuropeptides including crustacean cardioactive peptide, molt inhibiting hormone, L-enkephalin, FMRF-amide, proctolin, and crustacean hyperglycemic hormone precursor-related peptide were tested with regard to possible osmoregulatory roles with reference to changes in second messenger (cAMP and cGMP) concentrations in gill, hindgut, and midgut tissues in vitro, following application at 2 × 10 −8 M but all were found to be inactive. Thus, it seems likely that CHH is a pertinent neurohormone involved in osmoregulation, thus expanding its many functions as a pleiotropic hormone in crustaceans.

Effects of serotonin and fluoxetine on blood glucose regulation in two decapod species.

One of the best known crustacean hormones is the crustacean hyperglycemic hormone (CHH). However, the mechanisms involved in hormone release in these animals are poorly understood, and thus constitute the central objective of the present study. Different groups of crustaceans belonging to diverse taxa (Chasmagnathus granulata, a grapsid crab and Orconectes limosus, an astacid) were injected with serotonin, fluoxetine, or a mixture of both, and glycemic values (C. granulata and O. limosus) and CHH levels (O. limosus) were determined after 2 h in either submerged animals or animals exposed to atmospheric air. Both serotonin and fluoxetine caused significant hyperglycemia (P<0.05) after injection into the blood sinus of the two species, an effect enhanced after exposure to atmospheric air. In C. granulata blood glucose increased from 6.1 to 43.3 and 11.4 mg/100 ml in submerged animals and from 5.7 to 55.2 and 22.5 mg/100 ml in air-exposed animals after treatment with serotonin and fluoxetine, respectively. In O. limosus the increases were from 1.2 to 59.7 and 135.2 mg/100 ml in submerged animals and from 2.5 to 200.3 and 193.6 mg/100 ml in air-exposed animals after treatment with serotonin and fluoxetine, respectively. Serotonin and fluoxetine also caused a significant increase in the circulating levels of CHH in O. limosus, from 11.9 to 43 and 45.7 fmol/ml in submerged animals and from 13.2 to 32.6 and 45.7 fmol/ml in air-exposed animals, respectively, thus confirming their action as neuroregulators in these invertebrates.

Quantification of Stress in Lobsters: Crustacean Hyperglycemic Hormone, Stress Proteins, and Gene Expression

Various methods for the quantification of stress in crustaceans have been developed in our laboratory. An ELISA was developed for the crustacean hyperglycemic hormone (CHH) from the lobster, Homarus americanus . It is sensitive to as little as 0.2 fmol of peptide. Increases in hemolymph CHH were observed following emersion. Significant levels of hemolymph CHH were also measured in lobsters that had been eyestalk-ablated. It was observed that these animals continued to produce CHH, even though the heretofore only known source of CHH had been removed. Portions of the central nervous system, from both intact and eyestalk-ablated lobsters were observed to contain significant amounts of CHH. A cDNA library was constructed from eyestalk neural tissue of H. americanus . With the use of PCR, a 171 bp probe was isolated and purified. This probe was labeled and used to examine levels of CHH expression in the central nervous system (CNS) and in eyestalk neural tissue at different periods of the lobster molt cycle. CHH mRNA is present throughout the CNS. In the eyestalk, it is undetectable in postmolt, low in intermolt, and high in premolt. Stress proteins, also known as heat shock proteins (HSPs), are a highly conserved class of proteins which show elevated transcription during periods of stress in organisms as phylogenetically divergent as bacteria and humans. Using RT-PCR, we have partially cloned the lobster HSP90 gene. A 380 bp probe was 32P-labeled and hybridized with northern blots of midgut gland total RNA from heat-shocked lobsters. A 2 hr acute heat shock from 15%C (ambient water temperature) to 28%C resulted in a 6.0-fold induction of HSP90 after 6 hr of recovery at 15%C. A northern analysis of RNA isolated from the midgut glands of lobsters injected with 10 μg of the molting hormone 20-hydroxyecdysone displayed a 2.1-fold induction of HSP90 RNA 48 hr postinjection.

Quantification of Crustacean Hyperglycemic Hormone by ELISA in Hemolymph of the Lobster,Homarus americanus,Following Various Stresses

An ELISA was developed for the crustacean hyperglycemic hormone (CHH) from the lobster,Homarus americanus.It is sensitive to as little as 0.2 fmol of peptide. The assay was used to measure CHH in the hemolymph of intact lobsters after various environmental stresses. Increases in CHH were observed following emersion, exposure to high temperatures (23° and 28°C), and salinity stress (50 and 150% seawater). During emersion, concentrations of hemolymph glucose increased concomitantly with increases in CHH. Significant levels of hemolymph CHH were also measured in lobsters that had been eyestalk-ablated. These latter observations indicate that there may be a source of CHH other than the X-organ/sinus gland in the lobster.

Circadian Rhythmicity of the Crustacean Hyperglycemic Hormone (CHH) in the Hemolymph of the Crayfish.

The crustacean hyperglycemic hormone (CHH) is involved in the regulation of endogenous blood glucose metabolism. In this paper we describe the daily rhythmicity in the blood glucose and the blood CHH content of the crayfish Orconectes limosus. Both blood CHH and blood glucose levels increase during the first hours after the beginning of darkness. The bioactivity of released CHH is far higher than that of CHH stored in the sinus gland. Moreover, the released hyperglycemic material shows an affinity for high molecular weight proteins in the hemolymph. Preliminary results suggest that subunits of hemocyanin may act as potential carrier-proteins for bioactive CHH.