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.     

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.     

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.     

Opioid potentiated chromatophorotropin regulation of pigment migration in the land crab Gecarcinus lateralis

In Gecarcinus lateralis dopamine treatment results in dispersion of black and concentration of red pigments within chromatophores. These effects of dopamine on the migration of pigments can be blocked by the dopamine antagonist haloperidol. These results strongly indicate the presence of a dopamine receptor mediated system in this organism. Serotonin injections also result in the dispersion of black pigment; however, this effect cannot be blocked by haloperidol. Norepinephrine was found to be without effect on this pigment regulatory system. Injections of crude eyestalk extract results in pigment migration within the chromatophores in both stalked and destalked animals. Injection of the stable methionine enkephalin analog FK 33 824 into the organisms causes no observable effects on the pigment system. However, coinjection with eyestalk extract strongly potentiates the effect of the extract. This potentiation can be completely blocked by the opiate antagonist naloxone, thus indicating that an endogenous opioid system may be part of the overall regulation of pigmentation movement.     

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.     

Role of eyestalk hormone in the carbohydrate metabolism of a marine penaeid prawn,Parapenaeopsis hardwickii (MIERS) (Crustacea,Decapoda, Penaeidae)

Data furnished here concern with the role of eyestalk hormone in the regulation of carbohydrate metabolism in Parapenaeopsis hardwickii. Bilateral eyestalk ablation has brought about a significant (P < 0.01) fall and rise in the glycogen content in the midgut gland and abdominal muscle respectively. Although eyestalk ablation resulted in a significant (P < 0.01) depletion of fat in midgut gland, n0 significant (P > 0.05) change was observed in the abdominal muscle. Eyestalk extract administration in eyestalk-less prawns has significantly (P < 0.05) restored the glycogen and fat metabolites in the midgut gland. There was an obvious change in the glycogen content of the midgut gland and abdominal muscle of normal prawns when injected with eyestalk extracts from prawns in different molting stages. Eyestalk extract from intermolt prawns caused a significant (P < 0.05) decrease and increase in the glycogen quantity in the midgut gland and abdominal muscle respectively. Eyestalk extract from premolt and postmolt prawns has, although not significantly (P > 0.05), decreased and increased the utilization of glycogen respectively in the midgut gland. The physiological significance of these findings are discussed briefly.Paper forms part IV of the series

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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.     

The influence of optic nerve section on blood metabolite levels in Carcinus maenas (Crustacea: Brachyura)

• 1.1. Blood metabolite levels were assayed in Carcinus maenas as an indicator of the functioning of the hyperglycemic hormone, HGH, secreted by the crab's eyestalk neuroendocrine tissue.
• 2.2. Bilateral eyestalk ablation eventually resulted in a hypoglycemic response after 2–3 days.
• 3.3. Bilateral optic nerve section produced a significant, long-term hypoglycemic response suggesting that release of HGH from the eyestalk sinus gland is controlled, via a promotive neural pathway, by the CNS and probably by the cerebral ganglia.
• 4.4. Injection of eyestalk extract into operated crabs consistently produced significant, short-term hyperglycemia.

     

Bihormonal control of oogenesis in the freshwater prawn, Macrobrachium kistnensis

The hormonal control of oogenesis has been investigated in the freshwater prawn, Macrobrachium kistnensis. Experiments using bilateral eyestalk ablation and injection of eyestalk extract supported the presence of a gonad inhibiting factor in the eyestalk of female prawns. Injections of brain and thoracic ganglion extract in normal female prawns and in those subjected to bilateral eyestalk ablation revealed the presence of a gonad stimulating factor in the central nervous tissue.     

Influence of eyestalk removal on the histochemical distribution of oxidative enzymes in the cheliped muscle of Scylla serrata (Forskål)

Histochemical studies on the oxidative enzymes, NAD- and NADP-dependent isocitrate (IDH) and malate (MDH) dehydrogenases, succinic dehydrogenase (SDH), and cytochrome oxidase of the cheliped muscle of Scylla serrata (Forskål) indicated that their concentrations are relatively lower than those of vertebrate muscle. The site of action of various oxidative enzymes is found to be common in the component fibres varying in diameter. The sarcolemma generally exhibited stronger positive reactions for the enzymes than the sarcoplasm.The bilateral removal of eyestalks had a stimulatory effect on the activity of oxidative enzymes. Initially increased activity of SDH, IDH and MDH (NAD-linked) and cytochrome oxidase 2–4 h after eyestalk removal was found to be maintained after 24 h; a noticeable increase in the NADP-linked MDH was also apparent by this time.The eyestalk extract when injected into de-stalked animals, caused a decrease in the levels of SDH, NAD-linked IDH and MDH, and cytochrome oxidase. Biochemical estimations of SDH clearly indicate that bilateral eyestalk extirpation results in remarkably enhanced enzyme activity; conversely, the administration of eyestalk extract brings about a sharp decline in the enzyme concentration. Thus, it seems that the eyestalks may contain a factor regulating oxidative metabolism.     

Differential expression of CMG peptide and crustacean hyperglycemic hormones (CHHs) in the eyestalk of the giant tiger prawn Penaeus monodon

Mouse antiserum against C-terminal amide of Pem-CMG (a peptide in the family of CHH/MIH/GIH) penta-deca peptide (RPRQRNQYRAALQRLamide=CMG-15) was generated and used for localization of the peptide in tissue and extract of the eyestalk of Penaeus monodon by means of immunohistochemistry and dot-ELISA in comparison with anti-T+ antiserum (T+=YANAVQTVamide : the putative C-terminal amide of crustacean hyperglycemic hormone (CHH) of Macrobrachium rosenbergii). The anti-CMG-15 antiserum did not show cross-reactivity to T+ peptide by dot-ELISA and vice versa for anti-T+ antiserum. In dot-ELISA of eyestalk extract of P. monodon after one step separation by RP-HPLC, anti-CMG-15 antiserum recognized different peptide fractions (F38-39) from those recognized by anti-T+ antiserum (F19, 40-41 and 47-51). Most of the T+ immunoreactive fractions (except F19) show higher hyperglycemic activity than the CMG immunoreactive fractions. In immunohistochemical localization, anti-CMG antiserum recognized only 2-3 neurons in medulla terminalis X-organ complex (MTXO) with long processes terminated in the sinus gland. The CMG-immunoreactive neurons were clearly distinct from CHH containing neurons situated in the same area. This evidence confirms the existing of CMG peptide which may play distinct roles from CHHs in hormonal regulation in P. monodon.     

Histochemical patterns of lipolytic enzymes of the hepatopancreas of Scylla serrata and their possible relation to eyestalk factor(s)

Of the lipolytic enzymes studied histochemically, lipase (Tween 80 specific enzyme) showed highest activity while non-specific esterases (Tween 60, α-naphthyl acetate, β-naphthyl acetate and 5-bromo-indoxyl acetate esterases) and Tween 20 and Tween 40 esterases exhibited medium and lowest activities respectively. The pattern of distribution of these enzymes was found to be variable among the various elements of the hepatopancreas. Lipase. Tween 20 and Tween 40 esterases were localized at the apical parts of the R-cell, F-cell cytoplasm, the B-cell vacuolar contents and the lumina of the hepatopancreatic acini. Non-specific esterases were primarily present in the cytoplasm of the R-cells, brush border of the tubules and connective tissue. A striking reduction in the lipase activity of the R- and B-cells was apparent 4 hours after the bilateral extirpation of eyestalks. Restoration of activity was observed 48 hours after the operation. On the other hand, a rise in the level of non-specific esterases was conspicuous 4 hours after the eyestalk amputation. This increased enzyme activity was restored to normal 24 hours after the operation. Administration of eyestalk extract into normal and de-stalked animals caused an increase in the lipase activity of the R- and B-cells. Surprisingly, the activity of non-specific esterases also enhanced considerably in the R-cell cytoplasm and connective tissue. The enhanced activity of lipase and non-specific was noted 4 hours after the treatment. From the present findings it appears that the eyestalk hormone(s) are directly involved in regulating the activity of the lipolytic enzymes. The physiological role of the F- and B-cells seems to be secretion and extrusion of lipase respectively. The R-cells and connective tissue appear to be associated with storage and mobilization of lipids.     

Comparative immunohistochemistry and cellular distribution of farnesoic acid O-methyltransferase in the shrimp and the crayfish

Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to methylfarnesoate (MF) by the mandibular organ (MO) of crustaceans. Here we report the cellular localization of FAMeT and radiochemical assay of endogenous FAMeT activity in shrimp (Metapenaeus ensis) and crayfish (Procambarus clarkii) tissues. As in the eyestalk (ES), FAMeT is concentrated in specific neurosecretory cells of the ventral nerve cord (VNC) whereas only weak FAMeT immunoreactivity was observed in the MO. FAMeT was also detected in the ventral nerve cord, heart (HET), eyestalk, and muscle of the juvenile shrimp. Although the VNC shows the greatest FAMeT immunoreactivity, the heart extract exhibited the highest FAMeT enzymatic activity. These results suggest that FAMeT in the VNC may be inactive or inactivated at the stages of development tested. Contrary to the previous reports in other crustaceans, MO extract in shrimp shows only low FAMeT activity. The eyestalk, epidermis, ovary and testis show appreciable FAMeT activity. The presence of FAMeT in neurosecretory cells of VNC and eyestalk of shrimp and crayfish implies a possible interaction of FAMeT with the eyestalk CHH-family of neuropeptides. The widespread activity of FAMeT suggests that it has a wide spectrum of action in many tissues that contribute to the function and regulation of MF synthesis in shrimp and crayfish.     

Role of methionine-enkephalin on the regulation of carbohydrate metabolism in the rice field crab Oziotelphusa senex senex

In the present study, the role of eyestalks and involvement of methionine-enkephalin in the regulation of haemolymph sugar level was studied. Bilateral eyestalk ablation significantly decreased the haemolymph sugar levels, whereas injection of eyestalk extract into ablated crabs significantly increased the haemolymph sugar levels. Total carbohydrate (TCHO) and glycogen levels were significantly increased in hepatopancreas and muscle of eyestalk-ablated crabs, with a decrease in phosphorylase activity. Injection of eyestalk extract into ablated crabs resulted in partial/complete reversal of these changes. Injection of methionine-enkephalin into intact crabs significantly increased the haemolymph sugar level in a dose-dependent manner. Total tissue carbohydrate and glycogen levels were significantly decreased, with an increase in phosphorylase activity in hepatopancreas and muscle tissues of intact crabs after methionine-enkephalin injection. Methionine-enkephalin injection did not cause any changes in haemolymph sugar, tissue total carbohydrate and glycogen levels and activity levels of phosphorylase in eyestalk-ablated crabs. These results suggest that the eyestalks are the main source of hyperglycaemic hormone and methionine-enkephalin induces hyperglycaemia through eyestalks.     

Effect of x-organ sinus gland extract on [(35)S] methionine incorporation to the ovary of the red swamp crawfish Procambarus clarkii

The presence of gonad-inhibiting hormone in the x-organ sinus gland complex was evaluated in female Procambarus clarkii. Elimination of gonad-inhibiting hormone by way of eyestalk removal resulted in a large acceleration of ovarian development. Daily injection of four sinus gland equivalents reduced ovarian growth of eyestalk-ablated females by about 50% on day 6. Use of the radiotracer [(35)S] methionine showed that gonad-inhibiting activity reached its peak effect between 12 and 24 h following sinus gland injection. Dose-response showed that at least two sinus gland equivalents were needed to significantly counter the accelerated growth induced by eyestalk ablation. The high dose of extract needed to cause significant inhibition was attributed to this delayed response, which subsequently may have required a relatively prolonged exposure to the hormone.     

Vitellogenesis stimulated by thoracic ganglion implants into destalked immature spider crabs, Libinia emarginata.

Since destalked immature female Libinia emarginata fail to molt to maturity (Hinsch, 1972) and implants of thoracic ganglion of adult females into young females of Potamon dehaani caused a doubling of ovarian weight and differentiation of yolk granules (Otsu, 1963), the effects of the thoracic ganglion implantation on sexual maturation in Libinia was investigated. Destalked immature Libinia with a maximum carapace length of 4-5 cm received two implants of adult female thoracic ganglion at 10-day intervals. All of the surviving experimental animals and destalked controls molted to immaturity. Most of the experimental animals failed to complete a successful molt and few survived longer than 3-4 days. The experimental animals that survived for over a week showed signs of yolk deposition not observed in the destalked controls. Eggs in several stages of vitellogenesis were scattered throughout the gonad in the animal showing the most pronounced effect. This was not observed in any of the destalked controls.     

Function and cellular localization of farnesoic acid O-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis.

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.     

Blood glucose in marine penaeid prawns: I. Neuroendocrine regulation in Parapenaeopsis hardwickii (Miers). (Crustacea,Decapoda, Penaeidae)

In the present investigation on femaleP. hardwickii, the blood glucose level in bilateral eyestalk extirpated, reinjected with ES extract as well as normal prawns, administered with ES, different concentrations of BR and ThG extracts, was estimated quantitatively after fifteen minutes and two hours. During all treatments there was an immediate hike in blood glucose level after fifteen minutes but after 24 hr. of eyestalk ablation the level does not differ significantly (P > 0.05) from controls. There was a significant (P < 0.01) enhancement in blood glucose level in the cauterized eyestalkless and normal prawns injected with ES extract after 2 hr. There was a considerable (93% level of confidence) decrease in blood glucose level of normal prawns after injecting higher concentrations of BR and ThG extracts. There was an obvious change in blood glucose level due to the endocrine hormones injected from other prawns of the related family. ES extracts ofP. stylifera produced a significant (P < 0.01) increase, whereas higher concentration of BR and ThG extracts caused considerable (92% level of confidence) decrease in blood glucose level after injecting in cauterized eyestalkless prawns. The extracts of the same endocrine centres fromMetapenaeus monocerus did not induce any significant (P > 0.05) alteration in blood glucose quantity ofP. hardwickii. Hence from the above findings it may be inferred that, the hormones produced by eyestalks (X organ-Sinus gland Complex) posses a hyperglycemic factor, whereas hormones released from BR and ThG might be having a property to cause hypoglycemia (?).     

Effects of sinus gland extract on mandibular organ size and methyl farnesoate synthesis in the crawfish

Eyestalk inhibition of farnesoic acid O-methyl transferase, which mediates the final step of methyl farnesoate synthesis in the mandibular organ of the crawfish Procambarus clarkii, was evaluated. Eyestalk removal caused a 20-100-fold increase in methyl transferase activity 8-12 days following surgery. The surge in activity following eyestalk removal in males was approximately 4 days ahead of that of the females. This was accompanied by a three-fold increase in mandibular organ protein content. Methyl transferase inhibition was accomplished in vitro after only a 15-min exposure to sinus gland extract. The inhibition obtained by injecting animals in vivo was noticeably attenuated 6 h following injection. The contrast of the short-lived inhibition with the growth and prolonged increase following eyestalk removal suggests that the eyestalk exerts both chronic and acute effects on the mandibular organ.     

Effects of eyestalk ablation on growth and food conversion efficiency of the freshwater prawn Macrobrachium lanchesteri (de Man)

Bilateral eyestalk ablation in the freshwater prawn Macrobrachium lanchesteri results in high mortality, while unilateral eyestalk ablated prawns exhibited a high survival rate. There was marked increase in the growth of bilateral eyestalk-ablated prawns (47.70 mg/prawn) as compared to those that were unilaterally ablated (19.19 mg/prawn).