Methionine-enkephalin induces hyperglycemia through eyestalk hormones in the estuarine crab Scylla serrata

The hypothesis is tested that methionine-enkephalin, a hormone produced in and released from eyestalk of crustaceans, produces hyperglycemia indirectly by stimulating the release of hyperglycemic hormone from the eyestalks. Injection of methionine-enkephalin leads to hyperglycemia and hyperglucosemia in the estuarine crab Scylla serrata in a dose-dependent manner. Decreases in total carbohydrate (TCHO) and glycogen levels of hepatopancreas and muscle with an increase in phosphorylase activity were also observed in intact crabs after methionine-enkephalin injection. Eyestalk ablation depressed hemolymph glucose (19%) and TCHO levels (22%), with an elevation of levels of TCHO and glycogen of hepatopancreas and muscle. Tissue phosphorylase activity decreased significantly during bilateral eyestalk ablation. Administration of methionine-enkephalin into eyestalkless crabs caused no significant alterations in these parameters when compared to eyestalk ablated crabs. These results support the hypothesis that methionine-enkephalin produces hyperglycemia in crustaceans by triggering release of hyperglycemic hormone from the eyestalks.     

Hyperglycaemic effects of 5-hydroxytryptamine and dopamine in the freshwater prawn, Macrobrachium malcolmsonii

The effects of 5-hydroxytryptamine (5-HT; serotonin) and dopamine (DA) on tissue carbohydrate metabolism and haemolymph glucose levels in the freshwater prawn, Macrobrachium malcolmsonii, were investigated. Injection of 5-HT and DA produced hyperglycaemia in a dose-dependent and time-dependent manner, with DA being more effective than 5-HT. Interestingly, 5-HT and DA induced hyperglycaemia only in intact prawns but not in bilaterally eyestalk-ablated individuals. Total carbohydrate (TCHO) and glycogen levels decreased and phosphorylase activity increased in the hepatopancreas and muscle of intact prawns after being injected with 5-HT or DA. However, bilateral eyestalk ablation decreased haemolymph glucose and tissue phosphorylase activity and increased TCHO and glycogen levels of the hepatopancreas and muscle. Injection of 5-HT or DA did not cause significant changes in these variables in eyestalk-ablated prawns. It is hypothesized that 5-HT and DA induce hyperglycaemia in prawns by stimulating the release of crustacean hyperglycaemic hormone (CHH) from the X-organ sinus gland (XO-SG) complex located in the eyestalk.     

Effect of melatonin on hemolymph glucose and lactate levels in the fiddler crab Uca pugilator

Melatonin was injected into intact and eyestalk-ablated fiddler crabs (Uca pugilator), and its effects on hemolymph glucose and lactate levels were studied. In intact crabs, glucose and lactate levels cycled simultaneously, with peaks occurring during early and late photophase. Melatonin caused a shift in the glucose and lactate cycles, with only one peak occurring closer to mid-photophase. In eyestalk-ablated animals, the glucose rhythmicity was lost; lactate cycled, but levels were significantly lower than in intact animals. Melatonin caused a delayed hyperglycemia in eyestalk-ablated animals, with concurrent but much lower increases in lactate. Overall, melatonin demonstrated delayed hyperglycemic effects that do not appear to be mediated solely via eyestalk factors such as crustacean hyperglycemic hormone (CHH), though involvement of the eyestalks cannot be ruled out. An influence on extra-eyestalk CHH secretion is a potential mechanism of melatonin activity.     

Diurnal rhythm in levels of haemolymph sugar and hyperglycaemic activity of eyestalk extract in the fresh water rice field crab (Oziotelphusa senex senex Fabricius)

The concentration of haemolymph sugar and the hyperglycaemic activity of eyestalk extract was measured six times (8, 12, 16, 20 and 4 h) over a 24-h period. The concentration of haemolymph sugar and hyperglycaemic activity of eyestalk extract was higher during the night (0 h through 8 h) than that noted in day time (12 h). The variations are closely related to the activity of the animal.     

Carbohydrate metabolism in the stick insect, Carausius morosus

Age-related changes in some parameters related to carbohydrate metabolism in the stick insect, Carausius morosus, were investigated during the 6th instar and up to day 37 of adult life. Total haemolymph carbohydrate concentration and the fat body glycogen content are low and may be related to the low activity of this insect. Trehalose constitutes about 75–80% of the total blood carbohydrate pool. During the moult, total blood carbohydrate, fat body glycogen and haemolymph volume, decrease while glycogen phosphorylase activity of the fat body is slightly activated. The effects are brought about mainly by reduced feeding activity, but may also be influenced by the shedding and replacement of the cuticle. During starvation, blood homeostasis is maintained at the expense of fat body glycogen via an activation of phosphorylase. During reproduction, although no dramatic changes in fat body glycogen levels occur, blood carbohydrates are maintained and fat body phosphorylase is slightly activated. The possibility is discussed that during moulting and reproduction, blood sugar homeostasis is maintained by a hormonal mechanism controlling glycogen phosphorylase. No circadian rhythm in any parameter investigated is observed.     

Histochemical observations on the occurrence of glycolytic and pentose phosphate cycle enzymes in the hepatopancreas and their possible relation to eyestalk factor(s) in the crab Scylla serrata (Forskal).

Histochemical studies were carried out on some of the glycolytic enzymes viz. phosphorylase, aldose, alpha-glycerophosphate dehydrogenase (alpha-GPDH) and lactic dehydrogenase (LDH) and a key enzyme of the pentose phosphatase cycle, glucose-6-phosphate dehydrogenase (G-6-PDH), in the hepatopancreas of Scylla serrata (Forskal). 1. Weak activities of phosphorylase and aldolase and strong-activities of alpha-GPDH and LDH were noticed mainly in the brush border of the tubules and R-cell cytoplasm. A trace activity of G-6-PDH was noticed in the brush border. 2. Bilateral eyestalk removal results in inhibition of both phosphorylase and aldolase. However, enhanced activities of alpha-GPDH and LDH were noticeable 4 h after the operation. The G-6-PDH activity remained unaltered till 24 h. 3. Injection of eyestalk extract into both intact and destalked crabs activated all the enzymes.     

Melatonin and locomotor activity in the fiddler crab Uca pugilator

The influence of melatonin on locomotor activity levels was measured in the fiddler crab Uca pugilator. First, activity in untreated, laboratory-acclimated crabs was measured over 48 hours in a 12L:12D photoperiod; this study showed a nocturnal increase in activity. In eyestalk-ablated crabs, overall activity was significantly reduced, and no significant activity pattern occurred. Next, crabs were injected with melatonin or saline (controls) at various times during the 12L:12D photoperiod (0900h, 1200h, and twice at 2100h; each trial was separated by 3-4 days) and monitored for 3 hr post-injection. Control crabs had low activity during early photophase, high at mid-photophase, increasing activity during the first scotophase trial, and decreasing activity during the second scotophase trial. Melatonin had no significant influence on activity when injected during the early-photophase activity trough or early-scotophase activity decline, but significantly increased activity when injected during the mid-photophase activity peak and early-scotophase activity incline. Next, crabs were injected during an early scotophase activity trough and monitored throughout the twelve-hour scotophase. Melatonin did not increase activity until the mid-scotophase activity increase, approximately 6 hours later, showing that the pharmacological dosage persisted in the crabs' systems and had later effects during the incline and peak of activity but not the trough. Eyestalk-ablated crabs were injected with melatonin or saline during early photo- and scotophase. Melatonin significantly increased activity in the photophase but not the scotophase trial, indicating that the responsiveness to melatonin continues following eyestalk removal, but the timing may not match that of intact crabs. Melatonin may be involved in the transmission of environmental timing information from the eyestalks to locomotor centers in U. pugilator.     

On the mode of action of methionine enkephalin, FK 33-824 and naloxone in regulating the hemolymph glucose level in the fresh water field crab Oziotelphusa senex senex

The possible involvement of opioid system in the regulation of hemolymph glucose level in the fresh water crab Oziotelphusa senex senex Fabricius, was investigated. Opioid agonist and antagonist was also used in addition to methionine-enkephalin itself. Injection of the opioid, methionine-enkephalin and FK 33-824 significantly elevated hemolymph glucose level. In contrast, injection of naloxone in to crab resulted in decrease in hemolymph glucose level. Injection of naloxone prior to injection of methionine-enkephalin blocked the hyperglycemic action of methionine-enkephalin. Injection of methionine-enkephalin, FK 33824 and naloxone produced no significant effect on hemolymph glucose level in eyestalk-less crab. The alterations in the intact crab hemolymph glucose level hypothesised to be due to stimulation of release of hyperglycemic hormone during methionine-enkephalin and FK 33824 treatment and blocking of release of hyperglycemic hormone during naloxone treatment from the eyestalks of crab Oziotelphusa senex senex.     

Histochemical localization of oxidative enzymes in the hepatopancreas of scylla serrata (forskål) (brachyura: decapoda)

A histochemical study on the distribution of mitochondrial and oxidative enzymes viz., succinic, NAD and NADP-linked isocitrate and malic dehydrogenases and cytochrome oxidase in the hepatopancreas of Scylla serrata (Forskål) shows that the R cells possess the highest concentration of them at the apical parts; F and B cells showed poor staining reactions whereas E cells and connective tissue exhibited trace staining reactions. A moderate staining for these was obtained in the cells lining the main ducts.

The bilateral removal of eyestalks resulted in the stimulation of succinic and NAD-linked malic dehydrogenases and cytochrome oxidase, observed 4 h after the operation; after 24 h, however, these enzymes showed a slight reduction in the activity. A significant increase in the activity of NADP-linked isocitrate and malic dehydrogenases was noted 24 h after eyestalk ablation. The major alterations were noticed in the R cells.

After injecting eyestalk extract into destalked or intact crabs, a fall in the staining intensity of succinic, NAD-linked malic and isocitrate dehydrogenases in the R cells was apparent 2–4 h after the treatment but was subsequently re-established after 24 h.

It seems from the present results that there may be a factor(s) in the eyestalk of S. serrata which regulates the oxidative metabolism in the hepatopancreas. The physiological significance of oxidative enzymes in various cytologic elements is discussed.     

Regulation of neurohormone release in the fiddler crab, Uca pugilator: effects of gamma-aminobutyric acid, octopamine, Met-enkephalin, and beta-endorphin

Gamma-aminobutyric acid (GABA) blocked concentration of the pigments in melanophores and erythrophores of intact crabs. GABA blocked the release of pigment concentrating hormones from the isolated eyestalk. Octopamine (OA) blocked black pigment dispersion in intact crabs, but did not affect red pigment dispersion or concentration. OA blocked the release of black pigment dispersing hormone from isolated eyestalks. Met-enkephalin, but not Leu-enkephalin, stimulated black and red pigment concentration in intact crabs. Met-enkephalin, but not Leu-enkephalin, stimulated the release of pigment concentrating hormones from isolated eyestalks. Naloxone blocked the effects of Met-enkephalin in intact crabs and on isolated eyestalks. Beta-endorphin induced black pigment dispersion in intact crabs and in isolated legs.     

Development of imaginal competence to adipokinetic hormone in Locusta: lipid and carbohydrate levels, and glycogen phosphorylase activity in precocene-induced adultiforms

Abstract. Adipokinetic responses to injection of synthetic adipokinetic hormone I (sAKH) were investigated in precocene-induced fifth-instar adultiforms and in chemically allatectomized but morphogenetically normal adults of Locusta migratoria (L.). The results were compared with data on normal fifth (=last)-instar nymphs and normal adults. Chemical allatectomy did not affect the resting level of lipid in the haemolymph, nor the slight decrease of haemolymph carbohydrate concentration induced by sAKH. The effects of chemical allatectomy on sAKH-induced hyperlipaemia, the resting level of haemolymph carbohydrate, total glycogen phosphorylase specific activity, as well as sAKH-induced phosphorylase activation in the fat body, were mostly minor and probably indirect. The concentrations of lipid and carbohydrate in the haemolymph, and sAKH-induced activation of fat body glycogen phosphorylase were more or less similar in normal fifth-instar nymphs, normal adults and fifth-instar adultiforms. Thus, precocious metamorphosis did not affect markedly those parameters which show no or slight changes during normal metamorphosis. In contrast, parameters which change substantially during normal metamorphosis (sAKH-induced hyperlipaemia, sAKH-induced changes in haemolymph carbohydrate level and total glycogen phosphorylase activity) showed similar changes in the course of precocious metamorphosis; the values for fifth-instar adultiforms were similar to those obtained for normal adults, but differed markedly from those found in normal fifth-instar nymphs. Thus, accelerated (precocious) morphological adult development is strongly correlated with accelerated imaginal target competence to AKH and with relevant physiological development.     

Restricted specificity of a hyperglycaemic factor from the corpus cardiacum of the stick insect Carausius morosus

Extracts of the corpora cardiaca of the stick insect Carausius morosus elevate the haemolymph carbohydrate concentration in adult and 6th-instar larvae which are ligated behind the first pair of legs, but not in non-ligated (intact) insects. The increase in haemolymph sugars is due to trehalose elevation, is time dependent (with a maximal effect about 90–120 min after injection), and is dose dependent (needing 0.005 gland equivalents for a significant effect and a tenfold higher dose for a maximal response). The hyperglycaemic factor is localised entirely in the corpora cardiaca and appears to be specific to stick insects; corpora cardiaca extracts of two lepidopteran species (Acherontia atropos and Aglais urticae) and of Locusta migratoria have no effect, whereas corpora cardiaca extracts of the stick insects Cuniculina impigra and Sipyloidea sipylus have similar activity to those from C. morosus. This specificity is also shown when S. sipylus is used as the recipient. Synthetic adipokinetic hormone and red pigment concentrating hormone possess no hyperglycaemic activity in the stick-insect system. Two peaks of hyperglycaemic activity were obtained after column chromatography of corpora cardiaca extract of C. morosus on Sephadex G-25 and Sephadex LH-20. The factor seems to act via activation of fat-body glycogen phosphorylase, which, although 60% active in the control insects, is significantly increased to approx. 85% upon corpora cardiaca injection. However, the activation is demonstrated in ligated and intact insects. No significant decrease in the glycogen level of the fat body is observed after corpora cardiaca injection.     

In vivo regulation of the mandibular organ in the edible crab, Cancer pagurus

Considerable evidence indicates that methyl farnesoate (MF) production by the crustacean mandibular organs is negatively regulated by neuropeptides from the sinus gland (SG) in the eyestalk. In the crab Cancer pagurus, two neuropeptides (MO-IH-1 and -2) have been isolated from the SG that inhibit MF synthesis by mandibular organs of female crabs in vitro. To test their activity in vivo, we treated eyestalk-ablated male crabs with SG extracts (SGEs) or MO-IH-1 and -2. SGEs reduced haemolymph levels of MF by 60-80%, while MO-IH-1 and -2 had little effect. Protease treatment of SGEs destroyed the in vivo activity, suggesting that the extract contains an additional peptide responsible for the in vivo activity. When separated by reversed-phase high performance liquid chromatography (HPLC), the in vivo activity eluted in fractions prior to MO-IH-1 and -2. When mandibular organs were removed from animals previously treated in vivo with these active fractions, they had reduced levels of MF synthesis and activity of farnesoic acid O-methyl transferase compared with mandibular organs from animals treated with saline. Together, these results indicate that the regulation of the crustacean mandibular organ is complex and may involve several SG compounds. Some of these compounds (i.e., MO-IH-1 and -2) act directly on the tissue while others affect the mandibular organ indirectly.     

Development of imaginal competence to adipokinetic hormone in Locusta: Lipid and carbohydrate levels,and glycogen phosphorylase activity in azadirachtin-induced over-aged nymphs

• 1.1. Adipokinetic responses to injection of synthetic adipokinetic hormone I (sAKH) and relevant physiological parameters were studied in azadirachtin-induced 31 to 34-day-old Vth-instar over-aged male nymphs of Locusta migratoria which did not undergo the metamorphic moult to adult.
• 2.2. The resting lipid and carbohydrate levels in the haemolymph and sAKH-induced activation of fat body glycogen phosporylase did not differ markedly between over-aged nymphs, normal nymphs and normal adults.
• 3.3. Total fat body glycogen phosphorylase activity and the effect of sAKH on haemolymph carbohydrate level in over-aged nymphs were similar to those in normal adults and differed from those in normal nymphs.
• 4.4. sAKH-induced elevation of haemolymph lipid level in the over-aged nymphs was higher than in normal nymphs but lower than in normal adults; thus, the over-agad nymphs attained only a partial adult competence in this respect.

     

Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.     

Physiological compensation in unilateral eyestalk ablated crayfish, Cherax quadricarinatus

The eyestalks of crustaceans contain neurosecretory cells involved in the regulation of molting. In crayfish, bilateral ablation results in increased molting frequency and weight gain whereas unilateral ablation typically has no effect on molting frequency and weight gain. The effects of unilateral ablation were examined in juvenile Australian freshwater crayfish, Cherax quadricarinatus. As observed for other crayfish species, molting frequency and weight gain of unilateral ablated crayfish were not significantly different from control (intact) crayfish. Survival of unilateral ablated crayfish, however, was reduced compared to controls and was likely due to stress associated with the surgical procedure itself. Using radiolabeling techniques, protein synthesis was determined for neural tissues from the remaining eyestalk of ablated crayfish and compared to protein synthesis of neural tissues from eyestalks of control, non-ablated crayfish. Protein synthesis of ablated crayfish neural tissues was significantly higher (ca. 45%) than protein synthesis of control neural tissues. Electrophoretic analysis (SDS-PAGE and autoradiography) further demonstrated that protein synthesis increased linearly for all proteins in the remaining eyestalk of ablated crayfish. Together, these results suggest that a compensatory response occurred in unilateral ablated crayfish allowing normal physiological functions, particularly those involved in regulating growth cycles, to be maintained. J. Exp. Zool. 289:184-189, 2001.     

Histochemical localization of glycogen and some representative enzymes of glycolytic cycle in the cheliped muscle of Scylla serrata (Forskal) (Brachyura: Decapoda)

Glycogen, phosphorylase, aldolase, a-glycerophosphate dehydrogenase, lactic dehydrogenase and glucose-6-phosphate dehydrogenase were localized histochemically in the cheliped muscle of Scylla serrata (Forskal) in normal, destalked, destalked receiving eyestalk extract injection and intact injected with eyestalk extract.     

Variants in the histochemical patterns of lipids in the hepatopancreas of Scylla serrata (Forskal) (Brachyura, Decapoda).

The highest concentrations of phospholipid, neutral lipid and fatty acids were observed in the R cells and connective tissue of the hepatopancreas of Scylla serrata. The basal parts of the B cells and apical parts of the cells lining the main duct also showed moderate presence of these substances. The E cells however, except at their cell membranes were found to be devoid of lipids. F cells on the other hand exhibited lipoid complexes. Considerable reduction in the staining intensity of fatty acids were noticed 4 h after the bilateral ablation of eyestalks, neutral lipid undergo depletion 24 h after the operation whereas phospholipid reserves increase 48 h after the eyestalk removal. A fall in the quantity of neutral lipid and phospholipid was conspicuous when eyestalk extract was injected into normal or destalked crabs. From the present data it appears that R cells and connective tissue form major sites of lipid storage and in an intermolt animal eyestalk factor(s) may have an important role in the control of lipid metabolism.     

Metabolic effects of eyestalk removal in the crab Varuna litterata (Fabricius)

Metabolic effects of eyestalk removal in the crab V. litterata is studied. Bilateral eyestalk ablation results in hypoglycaemia and a fall and rise in the glycogen content of hepatopancreas and muscle respectively. Injection of unboiled eyestalk extract evokes hyperglycaemia, whereas boiled eyestalk extract does not produce any significant change in the glycaemia level. The other effect of eyestalk removal is a fall in the fat content of the hepatopancreas and these results are discussed.     

Circadian Locomotor Activity Rhythm in the Freshwater Crab Pseudothelphusa americana (De Saussure, 1857): Effect of Eyestalk Ablation

The circadian rhythm of locomotor activity in the freshwater crab, Pseudothelphusa americana, was studied in aquaria using infrared crossing sensors. Individuals with ablated eyestalks were compared with intact individuals in constant darkness (DD) and in light-dark cycles (LD). Our results showed that intact animals in DD displayed bimodal rhythms. In LD conditions the two peaks were associated with lights on and lights off, respectively. A significant difference in the free running periods before and after LD was observed in all intact animals. After eyestalk ablation (ES-X), the circadian rhythm of locomotor activity disappeared immediately, but reappeared several days later. Diurnal activity was seen in some ES-X animals when exposed to LD. Our results indicate that locomotor activity rhythm in P. americana is driven primarily by oscillators located outside the eyestalks, and that extraretinal photoreceptors mediate either entrainment or masking effects.