ISJ106R.PDF ISJ 2: 132-141, 2005 ISSN 1824-307X Review Hyperglycemic stress response in Crustacea S Lorenzon BRAIN Center, Department of Biology, University of Trieste, Italy Accepted September 27, 2005 Abstract Blood glucose level in crustaceans is controlled by the crustacean Hyperglycemic Hormone (cHH), released from the eyestalk neuroendocrine centres both under physiological and environmental stress conditions. Hyperglycemia is a typical response of many aquatic animals to pollutants and stress and, in crustaceans, increased circulating cHH and hyperglycemia are reported to result from exposure to several environmental stressors. Biogenic amines and enkephalin have been found to mediate the release of several neurohormones from crustacean neuroendocrine tissue and a model of the controlling network is proposed. Key words: Crustacea; glucose; crustacean Hyperglycemic Hormone (cHH); stress response; neuroendocrine control Introduction Hyperglycemia is a typical response of many aquatic animals to harmful physical and chemical environmental changes. In crustaceans increased circulating crustacean Hyperglycemic Hormone (cHH) titres and hyperglycemia are reported to occur following exposure to several environmental stressors (Durand et al., 2000; Lorenzon et al., 1997; 2002; Santos et al., 2001) in intact but not in eyestalkless animals (Fig.1), suggesting a cHH mediated response (Fingerman et al., 1981; Reddy and Bhagyalakshimi, 1994; Reddy et al., 1996; Lorenzon et al., 2000, 2004a). Toxicity induced by a pollutant is the result of interaction of the compound or one of its metabolites, with the biochemical events involved in the homeostatic control of a physiological process (Brouwer et al., 1990). Physiological processes are mostly coordinated by hormones. Anthropogenic chemicals can alter the hormonal (endocrine) systems of wildlife and the Corresponding Author: Simonetta Lorenzon BRAIN Center, Department of Biology, University of Trieste, via Giorgieri 7, I-34127 Trieste, Italy E-mail: lorenzon@units.it effects of organic and inorganic contaminants on functions regulated by hormones in crustaceans are being investigated with increased frequency because several of these phenomena could be used as biomarkers of environmental contamination. Heavy metals and organic compounds have been found to negatively affect hormonally-regulated functions, such as reproduction, molting, blood glucose level and pigmentary effectors in crustaceans (Fingerman et al., 1998; Depledge and Billinghurst, 1999). Therefore, biosentinel parameters and “early warning” of toxicity can be identified by looking for alterations in endocrine patterns (Fingerman et al., 1996). Neurosecretory structures in the eyestalk are the most important components of the neuroendocrine system of the stalk-eyed crustaceans. The hemolymph glucose concentration is mainly controlled by the cHH synthesized within the X-organ (XO) and released from the sinus gland (SG) complex in the eyestalk (Abramowitz et al., 1944; Fingerman, 1987). Biogenic amines and enkephalin (L/M-Enk) control the release of neurohormones from the crustacean neuroendocrine tissue. Serotonin (5-HT) is involved in regulating important aspects of behaviour and a variety of systemic physiological functions. 5-HT has long been known (Bauchau and Mengeot, 1966) to have a potent hyperglycemic effect in several crustacean species (Lorenzon et al., 1999, 2004b; Lee et al., 132 2000; Komali et al., 2005;), while dopamine (DA) and enkephalin showed conflicting results in different species (Sarojini et al., 1995; Lorenzon et al., 1999, 2004b; Zou et al., 2003; Komali et al., 2005). The crustacean Hyperglycemic Hormone (cHH) Multiple forms of the cHH represent one member of an eyestalk neuropeptide family (Bocking et al, 2001), that includes the moult inhibiting hormone (MIH) and the gonad inhibiting hormone (GIH): the cHH/MIH/GIH family. These neuropeptides, synthesized in the XO, a cluster of neuron perikarya located in the medulla terminalis of the eyestalk, are transported to and stored in the axon terminals forming a neurohemal organ named SG and released by exocytosis into the hemolymph (Fig. 2). The main function of cHH is the regulation of hemolymph sugar level: cHHs are also involved in other functions such as reproduction (De Kleijn et al., 1998; De Kleijn and van Herp, 1998), molting (Chung et al., 1999; Webster et al., 2000), lipid metabolism (Santos et al., 1997), stress response (Lorenzon et al., 1997; 2002;Chang et al., 1999; Durand et al., 2000; Santos et al., 2001) and hydromineral regulation (Spanings-Pierrot et al., 2000; Serrano et al., 2003). On the basis of the primary structure, the cHH/MIH/GIH family can be divided into two sub- families (De Kleijn et al., 1995; Lacombe et al., 1999): the cHH sub-family characterized by the cHH precursor-related peptide (CPRP) and the MIH/GIH sub-family without CPRP. The prepropeptide cHH consists of a signal peptide, CPRP and a peptide with 72–74 amino acids. Usually, the mature peptide has an amidated carboxyl terminus (De Kleijn and van Herp, 1998; Lacombe et al., 1999), which is important in conferring hyperglycemic activity in Penaeus japonicus as evidenced by bioassay of recombinant peptide (Katayama et al., 2003). In several crustacean species, different isoforms of cHH exist. In the American lobster Homarus americanus, cHH-A (8.583 Da) and cHH-B (8.638 Da) have been found, with different actions during the female biannual reproductive cycle (De Kleijn et al., 1995). Role of biogenic amines and enkephalin in blood glucose regulation Neurotransmitters such as 5-HT, DA and L/M-enk play a fundamental role in hormone modulation (Fingerman et al., 1994) and at the same time their level and functions can be altered by pollutants (Amiard-Triquet et al., 1986, Reddy et al., 1997). 5-HT is well known as a neurotransmitter in crustaceans on several grounds, and its levels have been measured in the nervous system and hemolymph of various crustacean species (Elofsson et al., 1982; Laxmyr, 1984; Kulkarni and Fingerman 1992), thus suggesting a possible role as a neurohormone (Rodriguez-Soza et al., 1997). In crustaceans 5HT is linked with discrete circuits that control movements of the foregut, escape behaviour, locomotion and posture as well as with higher-order behaviours such as aggression (Sosa et al., 2004). In addition 5-HT levels are sensitive to environmental stress. 5-HT has long been known to have a potent hyperglycemic effect in several crustacean species (Bauchau and Mengeot, 1966; Keller and Beyer 1968; Lüschen et al., 1993; Kuo et al., 1995; Santos et al., 2001). In our laboratory (Lorenzon et al., 1999, 2004b) we have demonstrated that 5-HT elevates blood glucose in Palaemon elegans, Astacus leptodactylus and Squilla mantis. However no such effects were found in eyestalkless individuals of these species, suggesting the involvement of the eyestalk hormone cHH in the hyperglycemic response. In all the species injection of the antagonist, ketanserin and CPH (cyproheptadine, 5-HT1 receptor inhibitor) were able to inhibit the hyperglycemic effect of 5-HT. 5-HT1 like receptors seemed to be more likely involved in mediating 5-HT action, as CPH was a more effective antagonist than ketanserin (5-HT2 receptor inhibitor and also putative DA antagonist). These data agree with those by Lee et al. (2000) in Procambarus clarkii suggesting that 5-HT induced hyperglycemia is mediated by 5-HT1 and 5-HT2 like receptors. Using ELISA very recently we have demonstrated in P. elegans that injection of 5-HT induced a rapid and massive release of cHH from the eyestalk into the hemolymph followed by hyperglycemia. On the contrary DA did not significantly affect cHH release and hyperglycemia (Lorenzon et al. 2005). DA and enkephalins showed conflicting results in different species (Table 1). Injection of DA induced marked decrease in blood glucose levels in P. elegans and S. mantis (Lorenzon et al., 1999, 2004b). On the other hand injection of the DA receptor blocker inhibits the effects on blood glucose, apparently allowing the release of cHH. These findings are in contrast with those by Lüschen et al., (1993) for Carcinus maenas, Kuo et al. (1995) for Penaeus monodon and Komali et al. (2005) for Macrobrachium malcolmsonii where DA induced hyperglycemia in intact animals. As for enkephalins, L/M-Enk elicited hypoglycemic response in intact S. mantis but not in eyestalkless individuals (Lorenzon et al. 2004a). These results confirm those of Jaros (1990), Lüschen et al. (1991), Rothe et al. (1991) and Sarojini et al. (1995) who reported that L/M-Enk induced hypoglycemia in Uca pugilator, C. maenas and P. clarkii respectively. On the other hand L-enk induced hyperglycemic response in intact but not in eyestalkless A. leptodactylus (Lorenzon et al. 2004). These observations are consistent with our previous findings in P. elegans (Lorenzon et al., 1999) and also with recent reports on Oziotelphusa senex senex (Reddy and Basha, 2001), on the mud crab Scylla serrata (Reddy and Kishori, 2001) and in the two prawns, Penaeus indicus and Metapenaeus monocerus (Kishori et al., 2001). In S. mantis injection of the opioid antagonist naloxone reversed the inhibitory effect on blood glucose of L-enk while in A. leptodactylus an additive effect on hyperglycemia was recorded (Lorenzon et al., 2004b). All these results corroborate the commonly held view that 5-HT, is a potent hyperglycemic effector and exerts its effect through cHH release from the 133 Fig.1 Stress response in Crustacea. medulla terminalis XO-sinus gland complex (MTXO- SG), mediated by modulation of electrical activity of XO cells (Saenz et al., 1997). A detailed reconstruction of the underlying neural circuitry suffers from lack of precise identification of neurosecretory cell types, contrasting results of electrophysiological evidence and discrepancies due to interspecific differences (Glowik et al., 1997; Saenz et al., 1997). Finally 5-HT appears to provide a major control mechanism for glucose mobilization whereas DA and L/M-enk act as modulators whose plasticity in use or actions varied among even closely related species. Stress response Stress induced by changes in environmental parameters, emersion, handling and transport during commercial processes requires homeostatic regulation that brings about behavioural and physiological alterations in aquatic animals. Hemolymph glucose concentration can change significantly with altered physiological and environmental conditions. Exposure to air during commercial transport and hypoxia are reported to induce hyperglycemia in many crustacean species like the spiny lobster, Jasus edwardsii (Morris and Oliver 1999; Speed et al., 2001), the crab, Eriocheir sinensis (Zou et al., 1996), the spider crab, Maia squinado (Durand et al., 2000) and the Norway lobster, Nephrops norvegicus (Spicer et al., 1990). Moreover hyperglycemia is reported in the giant prawn, Macrobrachium rosenbergii as a response to cold shock (Kuo and Yang, 1999). Blood glucose level increased in P. elegans and other crustacean species after injection of lipopolysaccharide (LPS) and the hyperglycemic effect, is likely mediated by the cHH since it does not occur in eyestalkless animals. It is dose-related and dependent on the different Gram negative bacterial LPS (Lorenzon et al., 1997, 2002). Heavy metals like Cd, Hg, and Cu cause hyperglycemia in the freshwater prawn, Macrobrachium kistenensis, the crab, Barytelphusa canicularis (Nagabhushanam and Kulkarni, 1981, Machele et al., 1989) and S. serrata (Reddy and Bhagyalakshmi, 1994). Moreover, CdCl2 induces hyperglycemia in intact crayfish P. clarkii, but not in the absence of the eyestalks, suggesting a cHH mediated response (Reddy et al., 1996). Our studies (Lorenzon et al., 2000) on the effect of heavy metals on blood glucose levels in P. elegans showed that the intermediate sublethal concentrations of Hg, Cd and Pb produced significant hyperglycemic responses while the highest concentrations elicited no hyperglycemia in 24 h. In contrast, animals exposed to Cu and Zn showed hyperglycemia even at high concentrations. This difference in response could be explained on the basis of the physiological roles these two essential 134 Fig. 2 General organization of neuroendocrine tissues in the eyestalk of crustaceans. elements play in crustaceans, and consequent tolerance adaptations, as opposed to the toxic, xenobiotic heavy metals Cd, Hg and Pb. On the other hand both groups of heavy metals failed to elicit a hyperglycemic responses in eyestalk ablated animals suggesting the involvement of MTXO-SG hormones, most likely cHH. However, in spite of the richness of information regarding variations in blood glucose levels following stress, much less is known about the stress-induced variation in cHH levels in the sinus gland and in the hemolymph. In the crayfish Orconectes limosus subjected to hypoxia, blood cHH titers raise within 15 min (Keller and Orth, 1990). In Cancer pagurus emersion induced an increase in the hemolymph cHH after 4 h (Webster, 1996). Using ELISA Chang et al. (1998) observed variation in the blood cHH in Homarus americanus following exposure to various environmental stresses. Emersion was found to be a potent stimulator of blood cHH while temperature and salinity variations were less effective. Moreover an increase in water temperature increased blood cHH in C. pagurus and P. clarckii (Wilcockson et al. 2002; Zou et al., 2003). In C. maenas it has been shown that the concentration of the cHH in the hemolymph increases dramatically during molting from 1-5 fmol 100µL-1 in the intermolt up to 150-200 fmol 100µL-1 during ecdysis (Chung et al., 1999). Variation in the hemolymph cHH titer were also observed in N. norvegicus infected by the parasitic dinoflagellate Hematodinium sp. (Stentiford et al., 2001). Using ELISA and bioassay tests we have recently demonstrated the relationship between an environmental stressor and the release of cHH from the eyestalk into the hemolymph and the hyperglycemic response in the shrimp, P. elegans (Lorenzon et al., 2004a). Moreover with this work we validated the use of a cross reactive antibody, anti- NencHH, to assess cHH level in the eyestalk and hemolymph of P. elegans. With the help of standard immunocytochemistry the antibody had previously been tested for recognition of cHH in the eyestalks of different species belonging to systematic groups increasingly remote in the phylogenetic tree: the decapods A. leptodactylus, N. norvegicus, P. elegans, Munida rugosa and the stomatopod S. mantis (Giulianini et al., 2002). Finally we have quantified the variations in the hemolymph cHH after a challenge with different stressors. In P. elegans exposure to copper induced a dose-related rapid and massive release of cHH from the eyestalk into the hemolymph at the higher, lethal concentration while a gradual and reduced discharge was observed at the lower concentration (Fig. 3). The relationship between exposure to a toxicant and release of the cHH was confirmed by variation in blood glucose with a dose related hyperglycemia that peaked 2 h after exposure to copper (Fig. 4). Animals exposed to sublethal concentrations of Hg showed similar quantitative and time course relations between toxicant, release of cHH from the eyestalk, increment of hormone level in the hemolymph and subsequent hyperglycemia as already described for copper contamination. Interestingly, however, the highest, lethal concentration induced the release of cHH from the eyestalk into the hemolymph but was not followed by a significant variation in blood glucose (Figs 5, 6). This situation could be related to the high toxicity of Hg which may interfere with the finer mechanisms that regulate hyperglycemic response. It is neither due to synaptic blockage of the superimposed neuronal release network (Lorenzon et al., 1999) nor limited release of circulating cHH as high levels of cHH are discharged from the SG into the hemolymph. It is not due to inhibition of peripheral receptors on glycogenolytic target organs: indeed native SG homogenate injected into eyestalkless shrimps exposed to lethal concentration of Hg for 3 h is still able to cause hyperglycemia (Lorenzon et al., 2000). High concentrations of Hg, instead, may change the functionality of the prepro-cHH processed during secretory steps and due to its ability to bind cysteines - six of which represent a highly conserved feature of the peptide structure (Lacombe et al., 1999) - Hg might alter the active configuration of the peptide, as seen in other systems (Rodgers et al., 2001), but not its immunoreactivity. Moreover Hg is known to impair osmoregulatory mechanisms in the crab, Eriocheir sinensis (Péqueux et al., 1996); and inhibit acetylcholinesterase activity in P. clarkii (Devi and Fingerman, 1995). The altered response in P. elegans exposed to high concentrations of Hg may also be related to physiological modifications induced by Hg at a different systemic level (Lorenzon et al., 2004a). Cu contamination induced variations of 5-HT of the eyestalk and hemolymph of P. elegans (Lorenzon et al., 2005). The release of 5-HT from the eyestalk appears to be very rapid and dose dependent. In the hemolymph 5-HT peak occurs after 30 min and again the concentration of circulating 5- HT is dose dependent. After 1 h the level of 5-HT slowly decreases to the basal level (Fig. 7). 135 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 A cH H ( p m o l S G e -1 ) Time (h) Cu ++ 0.1 mg L -1 Cu ++ 5 mg L -1 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 11 12 B cH H ( p m o l m L -1 ) Time (h) Cu ++ 0.1 mgL -1 Cu ++ 5 mgL -1 Fig. 3 Time course of cHH in the eyestalk homogenates (A) and in the hemolymph (B) of P. elegans after exposure to different concentrations of Cu++ and in relation to untreated controls. Values are expressed as means ± SD (n=4 repeated measures). 0,5 1 2 3 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 2,4 2,6 2,8 3,0 3,2 3,4 In cr e m e n t o f g ly ce m ia Time (h) Cu ++ 0.1 mg L -1 Cu ++ 5 mg L -1 Control Fig. 4 Time course of glycemia in the hemolymph of P. elegans after exposure to different concentrations of Cu++ and in relation to untreated controls. Values of increment given as: [(experimental value)/ (value displayed by the same animal at 0 h)]–1, are expressed means ± SD (N=10 repeated measures). The release of 5-HT from the eyestalk into the hemolymph after Cu exposure precedes in its time course the release of cHH, confirming its role as neurotransmitter acting on cHH neuroendocrine cells. The rapid and massive release of 5-HT from the eyestalk of individual species following exposure to Cu might have induced release of the cHH resulting in hyperglycemia in intact but not in eyestalkless animals. Lastly contamination with different doses of LPS, a bacterial thermostable endotoxin from E. coli, confirms the dose-related and convergent chain of events that leads to hyperglycemia. This suggests that blood glucose elevation is a general-purpose response to stressors and is likely to perform a protective role (Lorenzon et al., 2004a). Conclusion In spite of the vastness of information on hyperglcemic stress response in Crustacea, there still exist many questions. In the scheme presented in figure 8 a possible model of the controlling network is proposed. Stressors have been demonstrated to release the cHH and 5-HT from the eyestalk leading to an increase in their hemolymph concentrations. 5-HT exerts a positive influence inducing the release of cHH from the SG into the blood. The cHH then acts upon the target organs to release more than normal level glucose resulting in hyperglycemia. The DA 136 0,5 1 2 3 control 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 A cH H ( pm ol S G -1 ) Time (h) Hg ++ 0.1 mg L -1 Hg ++ 0.5 mg L -1 Hg ++ 5 mg L -1 control 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 11 12 BTime (h) cH H ( p m o l m L -1 ) Hg ++ 0.1 mg L -1 Hg ++ 0.5 mg L -1 Hg ++ 5 mg L -1 control Fig. 5 Time course of cHH in the eyestalk homogenates (A) and in the hemolymph (B) of P. elegans after exposure to three different concentrations of Hg++ and in relation to untreated control. Values are expressed means ± SD (N=4 repeated measures). 0,5 1 2 3 5 -0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 In cr e m e n t o f g ly ce m ia time (h) Hg ++ 0.1 mg L -1 Hg ++ 0.5 mg L -1 Hg ++ 5 mg L -1 control Fig. 6 Time course of glycemia in the hemolymph of P. elegans after exposure to three different concentrations of Hg++ and in relation to untreated controls. Values of increment given as: [(experimental value)/ (value displayed by the same animal at 0 h)]–1, are expressed means ± SD (N=10 repeated measures). receptor blocker, spiperone, inhibited the hypoglycemic action of DA and was found not to affect the ability of L/M-Enk to produce hypoglycemia. On the other hand, naloxone blocked the action of both L/M-Enk and DA, thereby allowing the release of cHH (Sarojini et al., 1995, Lorenzon et al., 1999). Apparently DA and L-enk produced hypoglycemia by inhibiting cHH release. These results suggest that in the chain of neurons terminating at the neuroendocrine cells that secrete cHH, dopaminergic neurons precede enkephalinergic neurons. We also suggest a role for the hemocytes in the hyperglycemic stress response as stressors affect both the total (THC) and the differential haemocyte count and that exocytosis of cHH granules from the eyestalk neuroendocrine cells can be elicited either by an early release from hemocytes of cytokines and/or other circulating messengers like 5-HT. Moreover LPS treated eyestalkless animals undergo less haemocytopenia than intact individuals. This suggests that previous cHH release and hyperglycemia can cause a decrease in THC, which eventually exerts a protective function (Lorenzon et al., 2002). In summary it may be said that indicators of stress responses are useful in assessing the short- term well-being or long-term health status of an animal (Fossi et al., 1997; Paterson and Spanoghe, 1997) and, such indicators have received considerable attention in commercially important species of decapod crustaceans (Paterson 137 0 0,5 1 2 3 control 0 5 10 15 20 25 30 35 40 A hemolymph Cu ++ 5 mg L -1 Cu ++ 0.1 mg L -1 Time(h) 5 -H T ( n g m L -1 ) 0 0,5 1 2 3 control 0 1 2 3 4 5 6 7 8 9 10 11 12 B eyestalk 5 -H T ( n g m L -1 ) Time (h) Cu ++ 5 mg L -1 Cu ++ 0.1 mg L -1 Fig. 7 Time course (0.5-3 h) of 5-HT in the hemolymph (A) and in the eyestalk (B) of P. elegans after exposure to different concentrations of Cu++ and in relation to untreated controls. Values are expressed means ± SD (n=4 repeated measures). Fig. 8 Possible model of hyperglycemic stress response controlling network. In the scheme: continuous arrow=demonstrated effect, dotted arrow= hypothesized effect, red arrow= stimulation, blue arrow= inhibition, green arrow=release. 138 and Spanoghe, 1997; Chang et al., 1999). A number of researchers have suggested different methods for quantifying the stress responses in crustaceans; which include the measurement of different hemocyte types in the hemolymph (Jussila et al., 1997 Lorenzon et al., 1999, 2001), and the physiological, biochemical (Paterson and Spanoghe, 1997; Stentiford et al.. 1999), and molecular changes in the tissues and the hemolymph (Fossi et al., 1997). Thus variations in the hemolymph glucose concentration in the hemolymph and of the cHH level in relation to stressors could be used as a tool to monitor a variety of stress responses. Acknowledgements The author is grateful to Prof. EA Ferrero and Dr. PG Giulianini for useful discussion and comments on the manuscript. The constructive comments of anonymous referees are kindly acknowledged. 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