Effect of 5-Hydroxytryptamine and Dopamine on the Carbohydrate Metabolism in the Shrimp, Penaeus monodon (Fabricius)

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1 World Journal of Fish and Marine Sciences 4 (6): 86-93, 1 ISSN IDOSI Publications, 1 DOI: 1.89/idosi.wjfms Effect of -Hydroxytryptamine and Dopamine on the Carbohydrate Metabolism in the Shrimp, Penaeus monodon (Fabricius) K. Nagur Babu, P.N. Pallavi, D.C. Reddy and V. Kalarani 1 Department of Fishery Science and Aquaculture, S.V. University, Tirupati-17, India Department of Biotechnology, Sri Padmavati Mahila Visvavidyalayam University, Tirupati- 17, India Abstract: Effect of -Hydroxytryptamine (-HT) and Dopamine (DA) on tissue carbohydrate metabolism, protein metabolism and haemolymph glucose levels were studied in the shrimp, Penaeus monodon. Dopamine was found to be more effective than serotonin. Serotonin and Dopamine induced hyperglycaemia only in intact prawns but not in eyestalk ablated individuals. Total carbohydrate and glycogen levels showed decrease and phosphorylase activity showed increase in the hepatopancreas and muscle of intact prawns after being injected with -HT/DA. However, eyestalk ablation recorded decreased haemolymph glucose and tissue phosphorylase activity and an increase in carbohydrate and glycogen levels in the hepatopancreas and muscle. At the same time total proteins, free amino acids and lipids recorded an increase in serotonin injected animals but decreased in dopamine treated animals compared to the controls. Key words: Penaeus monodon % -Hydroxytryptamine (-HT) % Dopamine (DA) % Carbohydrate Metabolism INTRODUCTION and serotonin in the crustacean nervous system is well established [6]. DA is widely distributed in the crustacean The tiger shrimp, Penaeus monodon, is one of the nervous system and has a diverse array of physiological most common penaeid shrimp species currently being effects as reviewed by Tierney [7]. DA has been reported cultured in the world. The culture of this species to stimulate the release of both the pigment-concentrating intensified from 1988 onwards in Taiwan and expanded hormone [8, 9] and the distal retinal pigment dark-adapting rapidly to other countries in Southeast Asia. It is known hormone [1] in the fiddler crab, Uca pugilator, which that rapidly degradable environments in intensive culture causes the release of crustacean hyperglycemic hormone ponds may result in stresses and increased incidences of from the X organ/ sinus gland complex of Orconectes disease and can lead to culture failure of shrimp crops. limosus, the shore crab, Carcinus maenas [11] and the Stress-induced neuroendocrine changes are thought to freshwater giant prawn, Macrobrachium rosenbergii [1] divert an organism's energy resources away from and to inhibit -hydroxytryptamine- stimulated testicular physiological functions such as reproduction, growth and maturation in the fiddler crab, U. pugilator [13] and certain immune processes to metabolic and behavioral ovarian maturation in U. pugilator [14], in P. clarkia [1] adaptations that may help the animal overcome a threat and in M. rosenbergii [16]. Studies on marine, freshwater and survive [1, ]. The effects of neuroregulators on the and terrestrial crustaceans have indicated that DA is physiological systems of cultured shrimp are of primary involved in ionic and osmotic regulation and such studies concern. Several biogenic amines which function were reviewed by Morris [17]. Previous studies indicated mainly as neuroregulators (i.e., neurotransmitters and that DA induces transient modulation of physiological neuromodulators), including serotonin, dopamine (DA), responses, depresses the immune ability and increases octopamine, histamine, noradrenaline (norepinephrine), susceptibility to Vibrio alginolyticus in white leg shrimp, adrenaline (epinephrine), tryptamine and tyramine, have Litopenaeus vannamei [18]. Li et al. [19] also reported been identified and quantitatively measured in crayfish, that DA depresses the immune ability and increases Pacifastacus leniusculus and other decapod crustaceans susceptibility to Lactococcus garvieae in the freshwater [3-]. Among the biogenic amines, the presence of DA giant prawn, M. rosenbergii. Corresponding Author: P.N. Pallavi, Department of Fishery Science and Aquaculture, S.V. University, Tirupati-17, India. 86

2 World J. Fish & Marine Sci., 4 (6): 86-93, 1 In crustaceans, biogenic amines function mainly as the coax of the second pair of walking legs respectively, neurotransmitters and Neuromodulators in the nervous in a 1µl volume for TCHO, glycogen and phosphorylase system, with some molecules serving as circulating experiments. Both eyestalks were ablated from all the neurohormones. Neuroregulators are compounds that prawns in groups 4. The eyestalks were extirpated by function either as neurotransmitters by acting on the cutting them off at the base without prior ligation with transfer of information between a neuron and an adjacent postoperative cautery of the wound. The eyestalkless target cell or as neuromodulators by amplifying or prawns were used for bioassay tests two days after dampening neurotransmitter activity. ablation to enable metabolism of circulating This paper aims at studying the effects of -HT and hyperglycaemic hormone. Haemolymph was collected DA on haemolymph glucose levels, carbohydrate through the arthrodial membrane at the base of a walking metabolism and protein metabolism of a marine prawn leg using a hypodermic syringe. The other tissues Penaeus monodon and testing the hypothesis that -HT (hepatopancreas and muscle) were then quickly dissected and DA produce hyperglycaemia in Penaeus monodon by on ice, weighed and used for further biochemical analysis. stimulating the release of hyperglycaemic hormone from XO-SG. -HT is a biogenic amine neurotransmitter found Haemolymph Glucose Level: Haemolymph (1µl) was in both vertebrates and invertebrates that affects a wide mixed with 3µl of 9% ethanol. After deproteinization variety of physiological and behavioral functions, and centrifugation (4 C, 14,g, 1min) the supernatant including reproduction, sleep, appetite, learning, pain was combined with a mixture of glucose enzyme reagent perception and circadian rhythm., serotonin has been (glucose -6-phosphate dehydrogenase and NADP) and shown to stimulate the release of several neurohormones colour reagents phenazine methosulphate and such as crustacean hyperglycemic hormone, red pigment idonitrotetrazolium chloride (loba chem., India) (procured dispersing hormone neurodeprassing hormone and from sigma, USA). After 3min the intensity of the colour molt- inhibiting hormone. was measured at 49nm and quantified against standards. MATERIALS AND METHODS Tissue TCHO and Glycogen Levels: TCHO concentration in the hepatopancreas and muscle were estimated in 1% Experimental Protocol: P.monodon juveniles (14g) were trichloroacetic acid (TCA) supernatant (4%w/v) and obtained from a commercial farm in Muttembaka village glycogen concentration was measured using the ethanolic near Nellore andra Pradesh and were transferred to the precipitate of TCA supernatant following the method of laboratory in aerated plastic containers and maintained in Carroll et al. [1]. To. ml clear supernatant,.ml of laboratory holding tanks for a week in continuously anthronine reagent was added and the contents were aerated and filtered marine water at 8±1 C with a 1hr boiled for 1min in a water bath. The samples were light-dark cycle. During this period the prawns were fed immediately cooled at room temperature. A standard with add libitum with commercial pelleted feed (CP Aquaculture a known amount of glucose was always tested along with India Ltd., Chennai, India) once a day after changing at the experimental samples. Absorbance was measured at least % of the ambient medium. A constant biomass: 6nm against a reagent blank. water volume ratio (1g/1) was maintained throughout. Feeding was stopped 4hr before the start of experiments Tissue Phosphorylase Activity: Phosphorylase Activity and no food was given during experimentation. The in the hepatopancreas and muscle was estimated by biogenic amines, -HT and DA, were obtained as colorimetric determination of inorganic phosphate hydrochlorides from sigma chemical Co. (St. Louis, Mo). released from glucose-1-phosphate (G-1-P) following the All test solutions were prepared afresh in degassed method of Cori et al. []. Tissue homogenate (%w/v) crustacean saline [] before the start of each experiment was prepared in an aqueous medium containing.1m to avoid oxidation. The biogenic amines were tested in sodium fluoride and.37m ethylenediamine tetra concentrations ranging 1G mol/animal in a volume of acetic acid (EDTA) ph6.8 and centrifuged at 3, rpm for 1µl. Prawns to be used in the experiments were divided 1 min. The supernatant, diluted to four times (1:3) with in to four groups, with each group consisting of six cysteine hydrochloride (.3M) sodium glycerophosphate individuals. The first group received no treatment served (.7M) buffer (ph 6.8), is used as an enzyme source. as control. The group and 3 independently received Initially.4 ml of enzyme was incubated with.mg of injections of -HT and DA (1G mol) through the base of glycogen for min at 3 C. The reaction was then 87

3 World J. Fish & Marine Sci., 4 (6): 86-93, 1 initiated by the addition of.ml of.16m G-1-P to one activity, glycogen, Total Carbohydrates, Proteins, Free tube (phosphorylase a) and a mixture of.ml G-1-P and amino acids, lipids and Free fatty acids in.4m adenosine--monophosphate to an other tube Hepatopancrease and Muscle of P.monodon were (phosphorylase ab). The activity of total phosphorylase studied. Injection of -HT and DA separately into intact (ab) and active phosphorylase (a) was measured after prawns induced significant dose-dependent incubating the reaction mixture for 1 and 3min, hyperglycaemia. It was also interesting to note that at this respectively. dose DA caused more hyperglycaemia than -HT in P.monodono. A time course for both -HT and DA- Proetien And Free Amino Acids Determination: Total induced hyperglycaemia at 1G mol/prawn showed protein and free amino acid concentration in enzyme clearly that haemolymph glucose levels increased source was determined by method of Lowry et al. [3] significantly within 3 min of -HT and DA injections and using bovine serum albumin (BSA) as standard and Free reached a peak at 9 min, to decline gradually thereafter. amino acids were determined by Moore and Stein [4] Hence a 9 min post-injection duration was selected to method. study changes in carbohydrate metabolism. Total carbohydrate(tcho) and glycogen Lipid and Fatty Acid Determination: Total lipid was concentration in the hepatopancreas and muscle of estimated by the method of Folch et al. [] and Free -HTand DA injected intact prawns were significantly Fatty acid content was estimated by the method of lower than those in the hepatopancreas and muscle of Natelson [6]. control prawns, suggesting metabolization of TCHO and glycogen and mobilization of glucose from the Statistical Analysis: A multiple comparisons (Duncan s) hepatopancreas and muscle to heamolymph. The results test was conducted to compare significant differences also showed that activity levels of phosphorylase a and among treatments using the SPSS computer software and ab significantly increased in both hepatopancreas and differences were considered significant when P<.1. muscle following -HT and DA injections. The ratio of active total phosphorylase also increased in both the RESULTS tissues after -HT and DA injections. Suggesting conversion of inactive phosphorylase to active Present results on the effect of injection of different phosphorylase. Interestingly, the magnitude of decreases concentrations of serotonin and dopamine on in TCHO and glycogen concentrations and increase in haemolymph glucose concentrations, Phasphorylase phosphorylase (a and ab) activity levels in both the Table 1: Effect of injection of - hydroxytryptamine (- HT) and dopamine (DA) (1G mol) on phosphorylase activity in the hepatopancreas of P. monodon Phrosphorylase activity (µ moles of inorganic phosphate formed / mg protein / hr) a ab a/ab (%) Control 4.3±. 6.6± ±.1*(-18.) 6.16±.1*(~ 13.8) HT- injected.63±.1 (33.) 7.7±.13 (16.6) ±.1*(- 44.) 6.6±.1*(- 1.). DA -injected.97±.11 (41.1) 7.89±.18 (19.1) ±.8*(-44.) 6.3±.11*(- 19.8).8 Table : Effect of injection of - hydroxytryptamine (- HT) and dopamine (DA) (1G mol) on phosphorylase activity in the muscle of P.monodon. Phrosphorylase activity (µ moles of inorganic phosphate formed / mg protein / hr) a ab a/ab (%) Control 6.3± ± ±.*(- 3.4) 7.4±.7*(- 16.3) 9.6 -HT- injected 7.67±.1 (3.1) 1.3±.16 (1.8) ±.*(- 3.) 7.77±.14* (-.) 64. DA -injected 8.3±.1 (8.8) 1.1±.17 (16.8) ±.7* (- 36.) 7.±.13* (-.3)

4 World J. Fish & Marine Sci., 4 (6): 86-93, 1 Glycogen (%) Total Carbohydrate (%) Control -HT DA Treatement Control -HT DA Fig. 1: Effect of injection of - hydroxytryptamine (- HT) Fig. 4: Effect of injection of - hydroxytryptamine (- HT) and dopamine (DA) 1G mol on glycogen and dopamine (DA) 1G mol on total levels levels in the muscle of and ablated carbohydrate(tcho) levels in the hepatopancras P. monodon. (Values, measured mean ± SD of six of intact and ablated P.monodon. (Values, individual observations) measured mean ± SD of six individual observation) Total Carbohydrate (%) TI Glucose (ml/dl) Glycogen (%) Control -HT DA Control -HT DA 3.87 Protein (%) Control -HT DA Fig. : Effect of injection of -hydroxytryptamine (- Fig. : Effect of injection of saline, -hydroxytryptamine HT) and dopamine (DA) 1G mol on total (-HT) and dopamine (DA) 1G mol on carbohydrate (TCHO) levels in the muscle of haemolymph glucose concentrations of intact and and ablated P. monodon. (Values, mean eyestalk-ablated P.monodon. (Values are mean ± ±SD of six individual observations) SD of six individual observations) Control -HT DA Fig. 3: Effect of injection of -hydroxytryptamine (- Fig. 6: Effect of injection of - hydroxytryptamine (- HT) HT) and dopamine (DA) 1G mol on and dopamine (DA) 1G mol on protein levels in glycogen levels in the hepatopancras of intact the hepatopancreas of intact and ablated and ablated P. monodon. (Values, measured mean P.monodon. (Values are measured mean ± SD of ± SD of six individual observations) six individual observations) HP Muscle 89

5 World J. Fish & Marine Sci., 4 (6): 86-93, 1 hepatopancreas and muscle was higher in DA injected 4 HP shrimps than in -HT injected shrimps. Bilateral eyestalk 4 Muscle ablation in shrimp caused a significant decrease in the 3 3 activity level of phosphorylase (a and ab) in the hepatopancreas and muscle, with concomitant significant increase in tissue TCHO, glycogen concentration, 1 proteins, free amino acids, lipids and triglycerides and a 1 significant decrease in haemolymph glucose concentration. Control -HT DA The results clearly demonstrate that bilateral eyestalk ablation caused a significant decrease (P<.1) in the Fig. 7: Effect of injection of - hydroxytryptamine (- HT) activity levels of phosphorylase ( a and ab ) in the and dopamine (DA) (1G mol) on lipid levels in the hepatopancreas and muscle of prawns, with a hepatopancreas of and concomitant increase in tissue TCHO and glycogen P.monodon. (Values are measured mean ± SD of concentration and a significant decrease in six individual observations) haemolymph glucose concentration. Interestingly 3 injection of both of serotonin and dopamine into eyestalk HP 3 ablated prawns did not cause any significant change in Muscle the activity levels of phosphorylase in the hepatopancreas and muscle composed to eyestalkless controls. Lipid (%) Free Amino Acid (%) Free Fatty Acid (%) Stress-induced neuroendocrine changes are thought Control -HT DA to divert an organism's energy resources away from physiological functions that do not immediately help the Fig. 8: Effect of injection of - hydroxytryptamine (- HT) animal overcome a threat and survive to those that do [1]. and dopamine (DA) 1G mol on free amino acid To meet the heightened energy demands of stressed levels in the Hepatopancras of intact and ablated animals, glycogen, due to its easy availability for energy P.monodon. (Values are measured mean ± SD of production, may be rapidly catabolized resulting in losses six individual observations) of those reserves in tissues; consequently resulting in a significant elevation in blood glucose levels. 14 HP Hyperglycemia as a secondary stress response has been Musle 1 documented in many species of crustacean in response to 1 a wide range of stresses [7, 1]. It is known that CHH, 8 synthesized and released from the X-organ sinus gland 6 complex in crustaceans, is an important neurohormone 4 involved in glucose metabolism. Hemolymph glucose levels significantly increased in shrimp P.monodon, that had received dopamine and Serotonin. The effects Control -HT DA appeared to have been dose dependent [8]. A similar trend of a hemolymph glucose increase was also observed Fig. 9: Effect of injection of - hydroxytryptamine (- HT) in shrimp which had received Norepinephrine. These facts and dopamine (DA) 1G mol on free fatty acid led us to hypothesize that stresses induce the release of levels in the Hepatopancras of intact and ablated Norepinephrine in the hemolymph and affect the target P.monodon. (Values are measured mean ± SD of tissue in L. vannamei, resulting in a hyperglycemic six individual observations) response. 9.6 DISCUSSION 9

6 World J. Fish & Marine Sci., 4 (6): 86-93, 1 The results presented in this study suggest that -HT free fatty acid levels. A significant increase in free amino and DA are involved in the regulation of carbohydrate acids (FAA) content could be due to degradation of metabolism in the marine water shrimp, P. monodon. A proteins. Total free amino acids (FAA) pool is very high significant increase in phosphorylase activity and in crustaceans and a part of this pool may be utilized to decrease in total carbohydrate (TCHO) and glycogen meet the energy demand [3]. Since proteins are said to be concentration in Hepatopancreas and muscle of P. the major components of energy source in crustaceans monodon followed by hyperglycemia indicate tissue [36], the amino acids being precursors of proteins, seem glycogen break down and subsequent mobilization of to be mobilized and converted into gluconeogenic sugar molecules from tissues to haemolymph. Similar precursors to meet extra energy requirements if needed. results have been obtained in the fresh water crab, oziotelphusa senex senex [9, 3] and estuarine crab, ACKNOWLEDGEMENTS Scylla serrata [31]. Although the eyestalk hormone that augments haemolymph glucose levels is conventionally The authors are grateful to the Prof. D.C. Reddy, called CHH, Hohnke and Scheer [3] suggested that the Head, Department of Fishery Science and Aquaculture, primary function of CHH was not to elevate haemolymph S.V. University, Tirupati for providing chemical and glucose level but to elevate intracellular glucose laboratory facilities and Financial assistance from the concentration through degradation of glycogen by University Grant Commission, New Delhi, is gratefully activation the enzyme phosphorylase. The conversion of acknowledgement. phosphorylase from its inactive to active form results in glycogen breakdown and the glycogen molecules thus REFERENCES produced might enter the heamolymph causing hyperglycaemia, a view supported by Telford [33] in 1. Maule, A.G. and S.P. Vanderkooi, Stresscrayfish. As described earlier, injection of -HTand DA in induced immune- endocrine interaction. In: P.H.M. to intact P.monodan might have stimulated the Balm, (Ed.), Stress Physiology in Animals. Academic phosphorylase system, causing degradation of glycogen, Press, Sheffield, UK, pp: -4. resulting in accumulation of sugar molecules in the. Ottaviani, E. and C. Franceschi, The tissues and concomitant release in to the haemolymph neuroendocrinology of stress from invertebrates to and activation of the phosphorylase system. In intact man. Prog. Neurobiol., 48: prawns injected separately with -HT and DA might occur 3. Kuo, C.M., C.R. Hsu and C.Y. Lin, 199. either by triggering the release of hyperglycaemic Hyperglycaemic effects of dopamine in tiger shrimp, hormone from XO-SG or by mimicking the action of this Penaeus monodon. Aquaculture, 13: hormone. Apparently as -HT and DA did not produce 4. Eloffson, R., L. Lazmyr, E. Rosengren and these changes in eyestalkess P.monodon. It seems most C. Hansson, 198. Identification and quantitative likely that these two biogenic amines produced the measurements of biogenic amines and dopamine in hyperglycaemic hormone from the XO-SG of eyestalks. the central nervous system and haemolymph of the The results that the magnitude of increase in the crayfish Pacifastacus lenuiusculus (Crustacea). hepatopancreatic total lipids was more in serotonin Comp. Biochem. Physiol., 71C: treated prawns than in dopamine treated ones. An. Fingerman, M. and R. Nagabhushnam, 199. increase in the total lipids as a result of release of GSH by Control of the release of crustacean hormones serotonin injection reflects the accumulation of vitellin by neuroregulators. Comp. Biochem. Physiol., during ovarian maturation. Similar results have been 1C: reported in P.semisulcatus for the lipid composition of 6. Fingerman, M., R. Nagabhushnam, R. Sarojini and purified vitellin [34]. This study reveals that serotonin P.S. Reddy, Biogenic amines in crustaceans: treatment might have enhanced ovarian maturation as a identification, localization and roles. J. Crust. Biol., result of increase in the hepatopancreatic lipid and free 14: fatty acids. However, in dopamine treated prawns, 7. Tierney, A.J., T. Kim and R. Abrams, 3. Dopamine inhibition of ovarian growth has been observed. This may in crayfish and other crustaceans: distribution in the be attributed to decrease in hepatopancreatic lipids, central nervous system and physiological functions. triglycerides, free amino acids, proteins and increase in Microsc. Res. Tech., 6:

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