- Email: [email protected]
Effect of serotonin on hemolymph glucose regulation in the fresh water edible crab Oziotelphusa senex senex
Aquaculture 266 (2007) 274 – 278 www.elsevier.com/locate/aqua-online
Effect of serotonin on hemolymph glucose regulation in the fresh water edible crab Oziotelphusa senex senex P. Sreenivasula Reddy ⁎, T. Pushpalatha Department of Biotechnology, Sri Venkateswara University, Tirupati - 517502, India Received 17 November 2006; received in revised form 12 February 2007; accepted 13 February 2007
Abstract 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Oziotelphusa; Eyestalks; Hyperglycemic hormone; Hemolymph glucose; Serotonin
1. Introduction In crustaceans, hemolymph glucose level is under the control of an eyestalk factor, called the crustacean hyperglycemic hormone (CHH). CHH was first reported as a diabetogenic factor by Abramowitz et al. (1944). They found that injection of eyestalk extracts of Uca pugilator induced significant hyperglycemia in Callinectes. This neuropeptide, synthesized in the X-organ, a cluster of neuron perikarya located in the medulla terminalis of the eyestalk, is transported to and stored in the axon terminals forming a neurohaemal organ named
⁎ Corresponding author. Tel.: +91 0877 2249320; fax: +91 0877 2249611. E-mail address: [email protected] (P. Sreenivasula Reddy). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.02.023
sinus gland and released by exocytosis into the hemolymph. The chemical nature and mode and site of action of CHH were established (see reviews Sedlmeier, 1985; Keller et al., 1985; Keller, 1992; Santos and Keller, 1993). The amino acid sequence of CHH peptide and nucleotide sequence of cloned CHH cDNA have been determined in several crustaceans (see reviews De Kleijn and Van Herp, 1995; Van Herp, 1998; Reddy and Ramamurthi, 1999; Lacombe et al., 1999; Chang, 2001). However, mechanisms involved in the release of CHH from eyestalks are poorly understood, and thus constitute the central objective of the present study. Neuroregulators are compounds that function either as neurotransmitters by acting on the transfer of information between a neuron and an adjacent target cell or as neuromodulators by amplifying neurotransmitter activity (Barchas et al., 1978). Biogenic amines function as neurotransmitters in a wide array of animals
P. Sreenivasula Reddy, T. Pushpalatha / Aquaculture 266 (2007) 274–278
275
including crustaceans (Fingerman, 1997). One such amine is serotonin (5-hydroxytryptamine). The levels of serotonin and localizations of serotonin-immunoreactive neurons have been determined biochemically and immunohistochemically in the nervous organs of several crustaceans (Livingstone et al., 1981; Elofsson, 1983; Laxmyr, 1984; Sandeman et al., 1988; Kulkarni and Fingerman, 1992; Rodriguez_Sosa et al., 1997; Escamilla-Chimal et al., 2001). Functionally, it is established that serotonin stimulates the release of several neurohormones in crustaceans (Keller and Beyer, 1968; Rao and Fingerman, 1975; Arechiga et al., 1985; Mattson and Spaziani, 1985). In crustaceans, serotonin (5-hydroxytryptamine) is one of the most experimentally studied neuroregulators and known to have a potent hyperglycemic effect (Lorenzon, 2005). It was hypothesized that serotonin exerts its hyperglycemic effect by enhancing the release of hyperglycemic hormone from the eyestalks and Lee et al. (2000) suggested that serotonininduced hyperglycemia is mediated by serotonin type 1 and type 2 receptors. As part of our research directed at the understanding of regulation of hormone release in crustaceans, in the present investigation the effect of injection of serotonin on regulation of hemolymph glucose levels in the fresh water edible crab Oziotelphusa senex senex was studied. Oziotelphusa senex senex is one of the important representatives of decapod crustacean and is a potential species for freshwater aquaculture. Though it is an edible crab and well known as ‘poorman's protein’, little is known about the physiology of the crab. Hence, in the present study an attempt was made to understand the involvement of serotonin in the hemolymph glucose regulation in the crab.
ligation but with cautery of the wound after operation. No mortalities were observed after operation and the crabs were used for experimentation 24 h after eyestalk ablation. Serotonin (5-hydroxytryptamina creatine sulfate) was purchased from Sigma Chemical Company (St Louis, MO) and dissolved in isoosmotic crab saline (Van Harreveld, 1936). Different doses (10− 12 to 10− 4 mol/crab) of serotonin were injected into crabs through the base of the chelae with a micro-syringe (Hamilton) in 10 μL volume. Crabs injected with 10 μL saline (Van Harreveld, 1936) served as controls. Hemolymph was collected 2 h after injection. Time-course effect of serotonin on hemolymph glucose level was determined in the crabs injected with 10− 6 mol serotonin/crab. Hemolymph was collected at different time-points (see Results) and used for glucose quantification. Different groups of animals were used for hemolymph collection at different time intervals. CHH levels were determined in the hemolymph of the crabs injected with 10− 6 mol serotonin/crab. Two hours after injection, hemolymph was collected and used for CHH assay. Hemolymph samples were collected from intact and experimental crabs through the arthrodial membrane of the coxa of the leg joint. Hemolymph samples collected from the same experiment were analyzed at the same time. Hemolymph glucose content was determined by the glucose oxidase assay kit (Sigma Chemical Co., St Louis, MO). The assay protocol essentially followed the one provided by the manufacturer. Hemolymph CHH content was determined using a sandwich-type ELISA (Santos and Keller, 1993; Webster, 1996; Chang et al., 1988).
2. Materials and methods
2.1. Glucose assay
Only intermolt (stage C4) male crabs, Oziotelphusa senex senex, with a body weight 30–32 g and carapace width 32–35 mm were used for the present study. The crabs were collected from paddy fields around Tirupati (Andhra Pradesh, India). The animals were maintained in the laboratory at 28 ± 1 °C and on a 12:12 h light:dark cycle for at least 10 days before being used in experiments. During this period they were fed with sheep meat daily ad libitum and ambient medium was changed 6 h after feeding. Feeding was stopped 24 h before the commencement of experiment to avoid changes in hemolymph glucose level due to prandial activity. For the present experiments, both intact and eyestalk-ablated crabs were used. Eyestalks were removed by cutting off the stalks at the base without prior
For the measurement of glucose, hemolymph (100 μL) was mixed with 300 μl of 95% ethanol and centrifuged at 12,000 g for 10 min at 4 °C. 100 μL of protein-free sample is mixed with a mixture of glucose enzyme reagent (glucose-6-phosphate dehydrogenase and NADP) and color reagent agents (phenazine methosulfate and iodonitrotetrazolium chloride) (kit from Sigma). After 30 min, the developed color was read at 490 nm. The data were quantified with standards. 2.2. ELISA Oziotelphusa senex senex CHH was purified from 300 sinus glands by means of HPLC (Reddy and Reddy, 2006) and injected into a rabbit to produce primary
276
P. Sreenivasula Reddy, T. Pushpalatha / Aquaculture 266 (2007) 274–278
Fig. 1. Dose-dependent effect of serotonin on the hemolymph glucose levels in intact (filled bars) eyestalk-ablated (ESX; open bars) O. senex senex. Each bar represents a mean ± SD of 10 individuals. Differing letters indicate significant differences (p b 0.001).
antibody. The IgG fraction was purified and used to coat the wells of a multiwell plate. Non-specific binding sites were blocked with assay buffer (2% bovine serum albumin, 0.03% EDTA, 0.02% NaN3 in phosphatebuffered saline (PBS; 136 mM NaCl, 10 mM Na2PO4, 2.5 mM KCl, 1.75 mM KH2PO4). The samples and standards were incubated overnight at 4 °C in moist, sealed containers. After washing five times with 300 μL PBS with 0.1% Tween 20, biotinylated second antibody was added and incubated for 5 h at 37 °C. The solution was then discarded and the wells were washed five times with PBS with 0.1% Tween 20. Streptavidin-peroxidase was added to each well and incubated for 1 h at 37 °C. After washing the wells, 100 μL of a 2,2′-azino-bis-3ethylbenz-thiazoline-6-sulfonic acid solution was added. The change in the absorbance at 405 nm was measured in a multiwell plate reader. Data were analyzed by one-way analysis of variance (ANOVA). Paired means were compared by the Student t-test. All analyses were performed using the SPSS version 10.0. The level of significance was set at p b 0.001.
For doses lower than 10− 9 mol/crab, however, serotonin did not elicit any hyperglycemic response, whereas doses higher than 10− 6 mol/crab exhibited a saturated response in inducing hyperglycemia. In the subsequent experiments, 10− 6 mol/crab was selected as injection dose. A time-course action of serotonin-induced hyperglycemia is presented in Fig. 2. The hemolymph glucose level increased significantly (p b 0.001) within 1-h after serotonin injection and reached a highest peak at 2-h. Hemolymph glucose level declined gradually after 2-h and reached control level 6-h post-injection (Fig. 2). In the next experiment we determined whether serotonin induced hyperglycemia was mediated by the
3. Results Injection of serotonin into intact crabs resulted in significant hyperglycemia in a dose-dependent manner when compared to the controls (Fig. 1), whereas injection of physiological saline did not cause any significant effect on hemolymph glucose level. At doses between 10− 9 mol/crab (61.85% increase from control) and 10− 6 mol/crab (221.87% increase from control), the effect was statistically significant and dose dependent.
Fig. 2. Time course of serotonin-induced hyperglycemia in O. senex senex. Each point represents a mean ± SD of 10 individuals. Values in parentheses represent % change from control (0 h).
P. Sreenivasula Reddy, T. Pushpalatha / Aquaculture 266 (2007) 274–278 Table 1 Effect of serotonin on hemolymph CHH levels in Oziotelphusa senex senex
277
eyestalk hormone, CHH. Eyestalkless crabs (24-h post ablation) were injected with different doses of serotonin. No elevation in hemolymph glucose concentration was observed 2-h after injection of serotonin in eyestalkablated crabs (Fig. 1). As expected, bilateral eyestalk ablation produced significant hypoglycemia (Fig. 1). The results indicate that serotonin-induced hyperglycemia required the presence of the eyestalks in Oziotelphusa. An effort was also made to determine whether CHH has a role in serotonin-induced hyperglycemia. Injection of serotonin produced significant (p b 0.001) increase in hemolymph CHH level (Table 1).
in the eyestalks (Saenz et al., 1997), even though the CHH producing cells were not specifically identified. Further Lee et al. (2000) presented data suggesting that serotonin-induced hyperglycemia in P. clarkii is mediated by type 1 and type 2 serotonin receptors. Recently, Lorenzon et al. (2005) demonstrated in Palaemon elegans that injection of serotonin induced a rapid and massive release of CHH from the eyestalks into the hemolymph followed by hyperglycemia. Our data also provide evidence that serotonin triggers the release of CHH from eyestalks into circulation. Increased circulating level of CHH after serotonin administration is also in agreement with the earlier results (Santos et al., 2001; Lorenzon et al., 2005). In summary, the results of the research provide the first evidence that serotonin-induced hyperglycemia in the fresh water crab Oziotelphusa senex senex is mediated by the release of CHH from the eyestalks into circulation. Based on these results experiments are being conducted to determine the effect of serotonin and selected serotonin releasers, serotonin blockers and serotonin potentiators on circulatory levels of other CHH family peptides in the crab.
4. Discussion
Acknowledgements
Bilateral eyestalk ablation resulted in significant hypoglycemia indicating the location of hyperglycemic hormone in the eyestalks of the crab, Oziotelphusa senex senex. This is in agreement with our earlier results (Reddy, 1999; Kishori et al., 2001; Reddy and Riaz Basha, 2001; Kishori and Reddy, 2005). Injection of serotonin induced significant hyperglycemia in Oziotelphusa senex senex in a dose-dependent manner. Serotonin induced hyperglycemia has been reported in the crab Carcinus maenas (Bauchau and Mangeot, 1966; Luschen et al., 1993) and in crayfishes Orconectes limosus (Keller and Beyer, 1968) and Procambarus clarkii (Lee et al., 2000). The results that serotonin-induced hyperglycemia only in intact crabs but not in eyestalk-ablated crabs are consistent with those obtained in O. limosus (Keller and Beyer, 1968) and P. clarkii (Lee et al., 2000). From the results it was hypothesized that serotonin acts by triggering the release of hyperglycemic hormone from the sinus gland of the eyestalks. This hypothesis is further supported by the finding of Strolenberg and Van Herp (1977) in the crayfish Astacus leptodactylus, serotonin increased the exocytotic activity of the sinus glands. It has also been reported that in crayfish P. clarkii, serotonin induced firing and enhanced bursting activity of a population of neurosecretory cells located
This study was sponsored by the grant from the Department of Science and Technology, New Delhi. The author wishes to express thanks to Head, Department of Biotechnology, S.V. University for providing laboratory facilities, to Prof. K.V.S. Sarma, Department of Statistics, S.V. University for statistical analysis of data. Mr. S. Umasankar maintained the animals in the laboratory.
Group
Hemolymph CHH concentration (fmol/mL)
Control Saline-injected Serotonin-injected
8.87 ± 3.25 9.08NS ± 4.12 (2.36) 41.63⁎ ± 13.48 (369.34)
Values are mean ± SD of 10 individuals. Values in the parentheses represent % change from control. ⁎p b 0.001; NS = not significant.
References Abramowitz, A.A., Hisaw, F.L., Papandrea, D.N., 1944. The occurrence of diabetogenic factor in the eyestalks of crustaceans. Biol. Bull. 86, 1–5. Arechiga, H., Flores, J., Garcia, U., 1985. Biosynthesis and release of the crustacean neurodepressing hormone. In: Lofts, B., Holmes, W.N. (Eds.), Current Trends in Comparative Endocrinology. Hong Kong University, Hong Kong, pp. 787–791. Barchas, J.D., Akil, H., Elliot, G.R., Holman, R.B., Watson, S.J., 1978. Behavioral neurochemistry: neuroregulators and behavioral states. Science 200, 964–973. Bauchau, A.G., Mangeot, J.C., 1966. Serotonine et glycemie chez les crustaces. Biol. Bull. 86, 1–5. Chang, E.S., 2001. Crustacean hyperglycemic hormone family: old paradigms and new perspectives. Am. Zool. 41, 380–388. Chang, E.S., Keller, R., Chang, S.A., 1988. Quantification of crustacean hyperglycemic hormone by ELISA in hemolymph of the lobster, Homarus americanus, following various stresses. Gen. Comp. Endocrinol. 111, 359–366.
278
P. Sreenivasula Reddy, T. Pushpalatha / Aquaculture 266 (2007) 274–278
De Kleijn, D.P.V., Van Herp, F., 1995. Molecular biology of neurohormonal precursors in the eyestalks of crustacean. Comp. Biochem. Physiol. 112, 573–579. Elofsson, R., 1983. 5-HT-like immunoreactivity in the central nervous system of the crayfish, Pacifastacus leniusculus. Cell Tissue Res. 232, 221–236. Escamilla-Chimal, E.G., Van Herp, F., Fanjul-Moles, M.L., 2001. Daily variations in crustacean hyperglycemic hormone and serotonin (5-HT) immunoreactivity during the development of crayfish. J. Exp. Biol. 204, 1073–1081. Fingerman, M., 1997. Crustacean endocrinology: a retrospective, prospective, and introspective analysis. Physiol. Zool. 70, 257–269. Keller, R., 1992. Crustacean neuropeptides: structures, functions and comparative aspects. Experientia 48, 439–448. Keller, R., Beyer, J., 1968. Zur hyperglykamischen Wirkung von Serotonin und Augenstielextrakt beim Flusskrebs Orconectes limosus. Z. Vgl. Physiol. 59, 78–85. Keller, R., Jaros, P.P., Kegel, G., 1985. Crustacean hyperglycemic neuropeptides. Am. Zool. 25, 207–211. Kishori, B., Reddy, P.S., 2005. Role of methionine-enkephalin on the regulation of carbohydrate metabolism in the rice field crab Oziotelphusa senex senex. C. R., Biol. 328, 812–820. Kishori, B., Premasheela, B., Ramamurthi, R., Ressy, P.S., 2001. Evidence for hyperglycemic effect of methionine-enkephalin in the prawns Penaeus indicus and Metapenaeus monocerus. Gen. Comp. Endocrinol. 123, 90–99. Kulkarni, G.K., Fingerman, M., 1992. Quantitative analysis by reverse phase high performance liquid chromatography of 5-hydroxytryptamine in the central nervous system of the red swamp crayfish, Procambarus clarkia. Biol. Bull. 182, 341–347. Lacombe, C., Greve, P., Martin, G., 1999. Overview on the subgrouping of the crustacean hyperglycemic hormone family. Neuropeptides 33, 71–80. Laxmyr, L., 1984. Biogenic amines and DOPA in the central nervous system of decapod crustaceans. Comp. Biochem. Physiol. 77C, 139–143. Lee, C.Y., Yau, S.M., Liau, C.S., Huang, W.J., 2000. Serotonergic regulation of blood glucose levels in the crayfish, Procambarus clarkii: site of action and receptor characterization. J. Exp. Zool. 286, 596–605. Livingstone, M.S., Schaeffer, S.F., Kravitz, E.A., 1981. Biochemistry and ultrastructure of serotonergic nerve endings in the lobster: serotonin and octopamine are contained in different nerve endings. J. Neurobiol. 12, 27–54. Lorenzon, S., 2005. Hyperglycemic stress response in crustacean. ISJ, Inf. Syst. J. 2, 132–141. Lorenzon, S., Edomi, P., Guilianini, P.G., Mettulio, R., Ferrero, E.A., 2005. Role of biogenic amines and cHH in the crustacean hyperglycemic stress response. J. Exp. Biol. 208, 3341–3347. Luschen, W., Willig, A., Jaros, P.P., 1993. The role of biogenic amines in the control of blood glucose level in the decapod crustacean, Carcinus maenas L. Comp. Biochem. Physiol. 105C, 291–296.
Mattson, M.P., Spaziani, E., 1985. 5-hydroxytryptamine mediates release of molt-inhibiting hormone activity from isolated crab eyestalk ganglia. Biol. Bull. 169, 246–255. Rao, K.R., Fingerman, M., 1975. Action of biogenic amines on crustacean chromatophores. IV. Analysis of the synergistic erythrophoric pigment dispersion evoked by 5-hydroxytryptamine and lysergic acid diethylamide in the dwarf crayfish Cambarellus shufeldti. Comp. Biochem. Physiol. 51C, 53–58. Reddy, P.S., 1999. A neurotransmitter role for methionine enkephalin in causing hyperglycemia in the fresh water crab Oziotelphusa senex senex. Curr. Sci. 76, 1126–1128. Reddy, P.S., Ramamurthi, R., 1999. Recent trends in crustacean endocrinology. PINSA 65, 15–32. Reddy, P.R., Reddy, P.S., 2006. Isolation of peptide hormones with pleotropic activities in fresh water crab Oziotelphusa senex senex. Aquaculture 259, 424–431. Reddy, P.S., Riaz Basha, Md., 2001. 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. Z. Naturforsch. 56C, 629–632. Rodriguez_Sosa, L., Picones, A., Rosete, G.C., Islas, S., Arechiga, H., 1997. Localization and release of 5-hydroxytryptamine in the crayfish eyestalk. J. Exp. Biol. 200, 3067–3077. Saenz, F., Garca, U., Arechiga, A., 1997. Modulation of electrical activity by 5-hydroxytryptamine in crayfish neurosecretory cells. J. Exp. Biol. 200, 3079–3090. Sandeman, D.C., Sandeman, R.E., Aitken, A.R., 1988. Atlas of serotonin containing neurons in the optic lobes and brain of the crayfish Cherax destructor. J. Comp. Neurol. 269, 465–478. Santos, E.A., Keller, R., 1993. Crustacean hyperglycemic hormone (CHH) and the regulation of carbohydrate metabolism: current perspectives. Comp. Biochem. Physiol. 106, 405–411. Santos, E.A., Keller, R., Rodriguez, E., Lopez, L., 2001. Effects of serotonin and fluoxetine on blood glucose regulation in two decapod species. Braz. J. Med. Biol. Res. 34, 75–80. Sedlmeier, D., 1985. Mode of action of crustacean hyperglycemic hormone. Am. Zool. 25, 223–232. Strolenberg, G.E.C., Van Herp, F., 1977. Mise en evidence du phenomene d'exocytose dans la glande du sinus d'Astacus leptodactylus (Nordmann) sous l'influence d'injections de serotonine. C. r. Acad. Sci. Paris 284D, 57–59. Van Harreveld, A., 1936. A physiological solution for fresh water crustaceans. Proc. Soc. Exp. Boil. Med. 34, 428–432. Van Herp, F., 1998. Molecular, cytological and physiological aspects the crustacean hyperglycemic hormone family. In: Coast, G.M., Webster, S.G. (Eds.), Recent Advances in Arthropod Endocrinology. Cambridge Univ. Press, Cambridge, pp. 53–70. Webster, S.G., 1996. Measurement of crustacean hyperglycemic hormone levels in the edible crab Cancer pagurus during emersion stress. J. Exp. Biol. 199, 1579–1585.
Effect of serotonin on hemolymph glucose regulation in the fresh water edible crab Oziotelphusa senex senex P. Sreenivasula Reddy ⁎, T. Pushpalatha Department of Biotechnology, Sri Venkateswara University, Tirupati - 517502, India Received 17 November 2006; received in revised form 12 February 2007; accepted 13 February 2007
Abstract 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Oziotelphusa; Eyestalks; Hyperglycemic hormone; Hemolymph glucose; Serotonin
1. Introduction In crustaceans, hemolymph glucose level is under the control of an eyestalk factor, called the crustacean hyperglycemic hormone (CHH). CHH was first reported as a diabetogenic factor by Abramowitz et al. (1944). They found that injection of eyestalk extracts of Uca pugilator induced significant hyperglycemia in Callinectes. This neuropeptide, synthesized in the X-organ, a cluster of neuron perikarya located in the medulla terminalis of the eyestalk, is transported to and stored in the axon terminals forming a neurohaemal organ named
⁎ Corresponding author. Tel.: +91 0877 2249320; fax: +91 0877 2249611. E-mail address: [email protected] (P. Sreenivasula Reddy). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.02.023
sinus gland and released by exocytosis into the hemolymph. The chemical nature and mode and site of action of CHH were established (see reviews Sedlmeier, 1985; Keller et al., 1985; Keller, 1992; Santos and Keller, 1993). The amino acid sequence of CHH peptide and nucleotide sequence of cloned CHH cDNA have been determined in several crustaceans (see reviews De Kleijn and Van Herp, 1995; Van Herp, 1998; Reddy and Ramamurthi, 1999; Lacombe et al., 1999; Chang, 2001). However, mechanisms involved in the release of CHH from eyestalks are poorly understood, and thus constitute the central objective of the present study. Neuroregulators are compounds that function either as neurotransmitters by acting on the transfer of information between a neuron and an adjacent target cell or as neuromodulators by amplifying neurotransmitter activity (Barchas et al., 1978). Biogenic amines function as neurotransmitters in a wide array of animals
P. Sreenivasula Reddy, T. Pushpalatha / Aquaculture 266 (2007) 274–278
275
including crustaceans (Fingerman, 1997). One such amine is serotonin (5-hydroxytryptamine). The levels of serotonin and localizations of serotonin-immunoreactive neurons have been determined biochemically and immunohistochemically in the nervous organs of several crustaceans (Livingstone et al., 1981; Elofsson, 1983; Laxmyr, 1984; Sandeman et al., 1988; Kulkarni and Fingerman, 1992; Rodriguez_Sosa et al., 1997; Escamilla-Chimal et al., 2001). Functionally, it is established that serotonin stimulates the release of several neurohormones in crustaceans (Keller and Beyer, 1968; Rao and Fingerman, 1975; Arechiga et al., 1985; Mattson and Spaziani, 1985). In crustaceans, serotonin (5-hydroxytryptamine) is one of the most experimentally studied neuroregulators and known to have a potent hyperglycemic effect (Lorenzon, 2005). It was hypothesized that serotonin exerts its hyperglycemic effect by enhancing the release of hyperglycemic hormone from the eyestalks and Lee et al. (2000) suggested that serotonininduced hyperglycemia is mediated by serotonin type 1 and type 2 receptors. As part of our research directed at the understanding of regulation of hormone release in crustaceans, in the present investigation the effect of injection of serotonin on regulation of hemolymph glucose levels in the fresh water edible crab Oziotelphusa senex senex was studied. Oziotelphusa senex senex is one of the important representatives of decapod crustacean and is a potential species for freshwater aquaculture. Though it is an edible crab and well known as ‘poorman's protein’, little is known about the physiology of the crab. Hence, in the present study an attempt was made to understand the involvement of serotonin in the hemolymph glucose regulation in the crab.
ligation but with cautery of the wound after operation. No mortalities were observed after operation and the crabs were used for experimentation 24 h after eyestalk ablation. Serotonin (5-hydroxytryptamina creatine sulfate) was purchased from Sigma Chemical Company (St Louis, MO) and dissolved in isoosmotic crab saline (Van Harreveld, 1936). Different doses (10− 12 to 10− 4 mol/crab) of serotonin were injected into crabs through the base of the chelae with a micro-syringe (Hamilton) in 10 μL volume. Crabs injected with 10 μL saline (Van Harreveld, 1936) served as controls. Hemolymph was collected 2 h after injection. Time-course effect of serotonin on hemolymph glucose level was determined in the crabs injected with 10− 6 mol serotonin/crab. Hemolymph was collected at different time-points (see Results) and used for glucose quantification. Different groups of animals were used for hemolymph collection at different time intervals. CHH levels were determined in the hemolymph of the crabs injected with 10− 6 mol serotonin/crab. Two hours after injection, hemolymph was collected and used for CHH assay. Hemolymph samples were collected from intact and experimental crabs through the arthrodial membrane of the coxa of the leg joint. Hemolymph samples collected from the same experiment were analyzed at the same time. Hemolymph glucose content was determined by the glucose oxidase assay kit (Sigma Chemical Co., St Louis, MO). The assay protocol essentially followed the one provided by the manufacturer. Hemolymph CHH content was determined using a sandwich-type ELISA (Santos and Keller, 1993; Webster, 1996; Chang et al., 1988).
2. Materials and methods
2.1. Glucose assay
Only intermolt (stage C4) male crabs, Oziotelphusa senex senex, with a body weight 30–32 g and carapace width 32–35 mm were used for the present study. The crabs were collected from paddy fields around Tirupati (Andhra Pradesh, India). The animals were maintained in the laboratory at 28 ± 1 °C and on a 12:12 h light:dark cycle for at least 10 days before being used in experiments. During this period they were fed with sheep meat daily ad libitum and ambient medium was changed 6 h after feeding. Feeding was stopped 24 h before the commencement of experiment to avoid changes in hemolymph glucose level due to prandial activity. For the present experiments, both intact and eyestalk-ablated crabs were used. Eyestalks were removed by cutting off the stalks at the base without prior
For the measurement of glucose, hemolymph (100 μL) was mixed with 300 μl of 95% ethanol and centrifuged at 12,000 g for 10 min at 4 °C. 100 μL of protein-free sample is mixed with a mixture of glucose enzyme reagent (glucose-6-phosphate dehydrogenase and NADP) and color reagent agents (phenazine methosulfate and iodonitrotetrazolium chloride) (kit from Sigma). After 30 min, the developed color was read at 490 nm. The data were quantified with standards. 2.2. ELISA Oziotelphusa senex senex CHH was purified from 300 sinus glands by means of HPLC (Reddy and Reddy, 2006) and injected into a rabbit to produce primary
276
P. Sreenivasula Reddy, T. Pushpalatha / Aquaculture 266 (2007) 274–278
Fig. 1. Dose-dependent effect of serotonin on the hemolymph glucose levels in intact (filled bars) eyestalk-ablated (ESX; open bars) O. senex senex. Each bar represents a mean ± SD of 10 individuals. Differing letters indicate significant differences (p b 0.001).
antibody. The IgG fraction was purified and used to coat the wells of a multiwell plate. Non-specific binding sites were blocked with assay buffer (2% bovine serum albumin, 0.03% EDTA, 0.02% NaN3 in phosphatebuffered saline (PBS; 136 mM NaCl, 10 mM Na2PO4, 2.5 mM KCl, 1.75 mM KH2PO4). The samples and standards were incubated overnight at 4 °C in moist, sealed containers. After washing five times with 300 μL PBS with 0.1% Tween 20, biotinylated second antibody was added and incubated for 5 h at 37 °C. The solution was then discarded and the wells were washed five times with PBS with 0.1% Tween 20. Streptavidin-peroxidase was added to each well and incubated for 1 h at 37 °C. After washing the wells, 100 μL of a 2,2′-azino-bis-3ethylbenz-thiazoline-6-sulfonic acid solution was added. The change in the absorbance at 405 nm was measured in a multiwell plate reader. Data were analyzed by one-way analysis of variance (ANOVA). Paired means were compared by the Student t-test. All analyses were performed using the SPSS version 10.0. The level of significance was set at p b 0.001.
For doses lower than 10− 9 mol/crab, however, serotonin did not elicit any hyperglycemic response, whereas doses higher than 10− 6 mol/crab exhibited a saturated response in inducing hyperglycemia. In the subsequent experiments, 10− 6 mol/crab was selected as injection dose. A time-course action of serotonin-induced hyperglycemia is presented in Fig. 2. The hemolymph glucose level increased significantly (p b 0.001) within 1-h after serotonin injection and reached a highest peak at 2-h. Hemolymph glucose level declined gradually after 2-h and reached control level 6-h post-injection (Fig. 2). In the next experiment we determined whether serotonin induced hyperglycemia was mediated by the
3. Results Injection of serotonin into intact crabs resulted in significant hyperglycemia in a dose-dependent manner when compared to the controls (Fig. 1), whereas injection of physiological saline did not cause any significant effect on hemolymph glucose level. At doses between 10− 9 mol/crab (61.85% increase from control) and 10− 6 mol/crab (221.87% increase from control), the effect was statistically significant and dose dependent.
Fig. 2. Time course of serotonin-induced hyperglycemia in O. senex senex. Each point represents a mean ± SD of 10 individuals. Values in parentheses represent % change from control (0 h).
P. Sreenivasula Reddy, T. Pushpalatha / Aquaculture 266 (2007) 274–278 Table 1 Effect of serotonin on hemolymph CHH levels in Oziotelphusa senex senex
277
eyestalk hormone, CHH. Eyestalkless crabs (24-h post ablation) were injected with different doses of serotonin. No elevation in hemolymph glucose concentration was observed 2-h after injection of serotonin in eyestalkablated crabs (Fig. 1). As expected, bilateral eyestalk ablation produced significant hypoglycemia (Fig. 1). The results indicate that serotonin-induced hyperglycemia required the presence of the eyestalks in Oziotelphusa. An effort was also made to determine whether CHH has a role in serotonin-induced hyperglycemia. Injection of serotonin produced significant (p b 0.001) increase in hemolymph CHH level (Table 1).
in the eyestalks (Saenz et al., 1997), even though the CHH producing cells were not specifically identified. Further Lee et al. (2000) presented data suggesting that serotonin-induced hyperglycemia in P. clarkii is mediated by type 1 and type 2 serotonin receptors. Recently, Lorenzon et al. (2005) demonstrated in Palaemon elegans that injection of serotonin induced a rapid and massive release of CHH from the eyestalks into the hemolymph followed by hyperglycemia. Our data also provide evidence that serotonin triggers the release of CHH from eyestalks into circulation. Increased circulating level of CHH after serotonin administration is also in agreement with the earlier results (Santos et al., 2001; Lorenzon et al., 2005). In summary, the results of the research provide the first evidence that serotonin-induced hyperglycemia in the fresh water crab Oziotelphusa senex senex is mediated by the release of CHH from the eyestalks into circulation. Based on these results experiments are being conducted to determine the effect of serotonin and selected serotonin releasers, serotonin blockers and serotonin potentiators on circulatory levels of other CHH family peptides in the crab.
4. Discussion
Acknowledgements
Bilateral eyestalk ablation resulted in significant hypoglycemia indicating the location of hyperglycemic hormone in the eyestalks of the crab, Oziotelphusa senex senex. This is in agreement with our earlier results (Reddy, 1999; Kishori et al., 2001; Reddy and Riaz Basha, 2001; Kishori and Reddy, 2005). Injection of serotonin induced significant hyperglycemia in Oziotelphusa senex senex in a dose-dependent manner. Serotonin induced hyperglycemia has been reported in the crab Carcinus maenas (Bauchau and Mangeot, 1966; Luschen et al., 1993) and in crayfishes Orconectes limosus (Keller and Beyer, 1968) and Procambarus clarkii (Lee et al., 2000). The results that serotonin-induced hyperglycemia only in intact crabs but not in eyestalk-ablated crabs are consistent with those obtained in O. limosus (Keller and Beyer, 1968) and P. clarkii (Lee et al., 2000). From the results it was hypothesized that serotonin acts by triggering the release of hyperglycemic hormone from the sinus gland of the eyestalks. This hypothesis is further supported by the finding of Strolenberg and Van Herp (1977) in the crayfish Astacus leptodactylus, serotonin increased the exocytotic activity of the sinus glands. It has also been reported that in crayfish P. clarkii, serotonin induced firing and enhanced bursting activity of a population of neurosecretory cells located
This study was sponsored by the grant from the Department of Science and Technology, New Delhi. The author wishes to express thanks to Head, Department of Biotechnology, S.V. University for providing laboratory facilities, to Prof. K.V.S. Sarma, Department of Statistics, S.V. University for statistical analysis of data. Mr. S. Umasankar maintained the animals in the laboratory.
Group
Hemolymph CHH concentration (fmol/mL)
Control Saline-injected Serotonin-injected
8.87 ± 3.25 9.08NS ± 4.12 (2.36) 41.63⁎ ± 13.48 (369.34)
Values are mean ± SD of 10 individuals. Values in the parentheses represent % change from control. ⁎p b 0.001; NS = not significant.
References Abramowitz, A.A., Hisaw, F.L., Papandrea, D.N., 1944. The occurrence of diabetogenic factor in the eyestalks of crustaceans. Biol. Bull. 86, 1–5. Arechiga, H., Flores, J., Garcia, U., 1985. Biosynthesis and release of the crustacean neurodepressing hormone. In: Lofts, B., Holmes, W.N. (Eds.), Current Trends in Comparative Endocrinology. Hong Kong University, Hong Kong, pp. 787–791. Barchas, J.D., Akil, H., Elliot, G.R., Holman, R.B., Watson, S.J., 1978. Behavioral neurochemistry: neuroregulators and behavioral states. Science 200, 964–973. Bauchau, A.G., Mangeot, J.C., 1966. Serotonine et glycemie chez les crustaces. Biol. Bull. 86, 1–5. Chang, E.S., 2001. Crustacean hyperglycemic hormone family: old paradigms and new perspectives. Am. Zool. 41, 380–388. Chang, E.S., Keller, R., Chang, S.A., 1988. Quantification of crustacean hyperglycemic hormone by ELISA in hemolymph of the lobster, Homarus americanus, following various stresses. Gen. Comp. Endocrinol. 111, 359–366.
278
P. Sreenivasula Reddy, T. Pushpalatha / Aquaculture 266 (2007) 274–278
De Kleijn, D.P.V., Van Herp, F., 1995. Molecular biology of neurohormonal precursors in the eyestalks of crustacean. Comp. Biochem. Physiol. 112, 573–579. Elofsson, R., 1983. 5-HT-like immunoreactivity in the central nervous system of the crayfish, Pacifastacus leniusculus. Cell Tissue Res. 232, 221–236. Escamilla-Chimal, E.G., Van Herp, F., Fanjul-Moles, M.L., 2001. Daily variations in crustacean hyperglycemic hormone and serotonin (5-HT) immunoreactivity during the development of crayfish. J. Exp. Biol. 204, 1073–1081. Fingerman, M., 1997. Crustacean endocrinology: a retrospective, prospective, and introspective analysis. Physiol. Zool. 70, 257–269. Keller, R., 1992. Crustacean neuropeptides: structures, functions and comparative aspects. Experientia 48, 439–448. Keller, R., Beyer, J., 1968. Zur hyperglykamischen Wirkung von Serotonin und Augenstielextrakt beim Flusskrebs Orconectes limosus. Z. Vgl. Physiol. 59, 78–85. Keller, R., Jaros, P.P., Kegel, G., 1985. Crustacean hyperglycemic neuropeptides. Am. Zool. 25, 207–211. Kishori, B., Reddy, P.S., 2005. Role of methionine-enkephalin on the regulation of carbohydrate metabolism in the rice field crab Oziotelphusa senex senex. C. R., Biol. 328, 812–820. Kishori, B., Premasheela, B., Ramamurthi, R., Ressy, P.S., 2001. Evidence for hyperglycemic effect of methionine-enkephalin in the prawns Penaeus indicus and Metapenaeus monocerus. Gen. Comp. Endocrinol. 123, 90–99. Kulkarni, G.K., Fingerman, M., 1992. Quantitative analysis by reverse phase high performance liquid chromatography of 5-hydroxytryptamine in the central nervous system of the red swamp crayfish, Procambarus clarkia. Biol. Bull. 182, 341–347. Lacombe, C., Greve, P., Martin, G., 1999. Overview on the subgrouping of the crustacean hyperglycemic hormone family. Neuropeptides 33, 71–80. Laxmyr, L., 1984. Biogenic amines and DOPA in the central nervous system of decapod crustaceans. Comp. Biochem. Physiol. 77C, 139–143. Lee, C.Y., Yau, S.M., Liau, C.S., Huang, W.J., 2000. Serotonergic regulation of blood glucose levels in the crayfish, Procambarus clarkii: site of action and receptor characterization. J. Exp. Zool. 286, 596–605. Livingstone, M.S., Schaeffer, S.F., Kravitz, E.A., 1981. Biochemistry and ultrastructure of serotonergic nerve endings in the lobster: serotonin and octopamine are contained in different nerve endings. J. Neurobiol. 12, 27–54. Lorenzon, S., 2005. Hyperglycemic stress response in crustacean. ISJ, Inf. Syst. J. 2, 132–141. Lorenzon, S., Edomi, P., Guilianini, P.G., Mettulio, R., Ferrero, E.A., 2005. Role of biogenic amines and cHH in the crustacean hyperglycemic stress response. J. Exp. Biol. 208, 3341–3347. Luschen, W., Willig, A., Jaros, P.P., 1993. The role of biogenic amines in the control of blood glucose level in the decapod crustacean, Carcinus maenas L. Comp. Biochem. Physiol. 105C, 291–296.
Mattson, M.P., Spaziani, E., 1985. 5-hydroxytryptamine mediates release of molt-inhibiting hormone activity from isolated crab eyestalk ganglia. Biol. Bull. 169, 246–255. Rao, K.R., Fingerman, M., 1975. Action of biogenic amines on crustacean chromatophores. IV. Analysis of the synergistic erythrophoric pigment dispersion evoked by 5-hydroxytryptamine and lysergic acid diethylamide in the dwarf crayfish Cambarellus shufeldti. Comp. Biochem. Physiol. 51C, 53–58. Reddy, P.S., 1999. A neurotransmitter role for methionine enkephalin in causing hyperglycemia in the fresh water crab Oziotelphusa senex senex. Curr. Sci. 76, 1126–1128. Reddy, P.S., Ramamurthi, R., 1999. Recent trends in crustacean endocrinology. PINSA 65, 15–32. Reddy, P.R., Reddy, P.S., 2006. Isolation of peptide hormones with pleotropic activities in fresh water crab Oziotelphusa senex senex. Aquaculture 259, 424–431. Reddy, P.S., Riaz Basha, Md., 2001. 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. Z. Naturforsch. 56C, 629–632. Rodriguez_Sosa, L., Picones, A., Rosete, G.C., Islas, S., Arechiga, H., 1997. Localization and release of 5-hydroxytryptamine in the crayfish eyestalk. J. Exp. Biol. 200, 3067–3077. Saenz, F., Garca, U., Arechiga, A., 1997. Modulation of electrical activity by 5-hydroxytryptamine in crayfish neurosecretory cells. J. Exp. Biol. 200, 3079–3090. Sandeman, D.C., Sandeman, R.E., Aitken, A.R., 1988. Atlas of serotonin containing neurons in the optic lobes and brain of the crayfish Cherax destructor. J. Comp. Neurol. 269, 465–478. Santos, E.A., Keller, R., 1993. Crustacean hyperglycemic hormone (CHH) and the regulation of carbohydrate metabolism: current perspectives. Comp. Biochem. Physiol. 106, 405–411. Santos, E.A., Keller, R., Rodriguez, E., Lopez, L., 2001. Effects of serotonin and fluoxetine on blood glucose regulation in two decapod species. Braz. J. Med. Biol. Res. 34, 75–80. Sedlmeier, D., 1985. Mode of action of crustacean hyperglycemic hormone. Am. Zool. 25, 223–232. Strolenberg, G.E.C., Van Herp, F., 1977. Mise en evidence du phenomene d'exocytose dans la glande du sinus d'Astacus leptodactylus (Nordmann) sous l'influence d'injections de serotonine. C. r. Acad. Sci. Paris 284D, 57–59. Van Harreveld, A., 1936. A physiological solution for fresh water crustaceans. Proc. Soc. Exp. Boil. Med. 34, 428–432. Van Herp, F., 1998. Molecular, cytological and physiological aspects the crustacean hyperglycemic hormone family. In: Coast, G.M., Webster, S.G. (Eds.), Recent Advances in Arthropod Endocrinology. Cambridge Univ. Press, Cambridge, pp. 53–70. Webster, S.G., 1996. Measurement of crustacean hyperglycemic hormone levels in the edible crab Cancer pagurus during emersion stress. J. Exp. Biol. 199, 1579–1585.