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Methionine-Enkephalin Induces Hyperglycemia Through Eyestalk Hormones in the Estuarine Crab Scylla serrata.

P. SREENIVASULA REDDY [*]

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

Introduction

In decapod crustaceans, hemolymph sugar level is regulated by hyperglycemic hormone. Abramowitz et al. (1944) were the first to demonstrate that injection of eyestalk extract induced hyperglycemia in Callinectes. Since then, hyperglycemia as a response to injection of eyestalk extract has been observed in almost all groups of crustaceans (see review by Keller, 1992). This neurohormone is stored in and released from the sinus gland. The chemical nature, mode, and site of action of hyperglycemic hormone has been extensively studied in a number of crustaceans (see reviews by Keller et al., 1985; Sedlmeier, 1985). The amino acid sequence of hyperglycemic hormones has been determined from a large number of crustaceans (see La Combe et al., 1999, for review). The gene for hyperglycemic hormone was also cloned from crabs (Kegel et al., 1989), lobster (Tensen et al., 1991), prawn (Ohira et al., 1997), isopod (Martin et al., 1993), and crayfish (Kegel et al,, 1991; Huberman et al., 1993; Yasuda et al., 1994). Recentl y, we reported the expression of hyperglycemic hormone gene at different molt stages in Homarus americanus, the American lobster (Reddy et al., 1997).

Since the discovery of opioid peptides in decapod crustaceans by Mancillas et al. (1981), several workers have attempted to determine the physiological function of these peptides, but the results are fragmentary. Sarojini et al. (1995, 1996, 1997) provided evidence that methionineenkephalin slowed ovarian maturation in the fiddler crab Uca pugilator and the crayfish Procambarus clarkii, and suggested that methionine-enkephalin produces this effect indirectly by stimulating the release of gonad-inhibiting hormone from eyestalks. In Uca pugilator, methionine-enkephalin appears to stimulate release of the concentrating hormones for black and red pigment cells (Quackenbush and Fingerman, 1984) and the dark-adapting hormone for distal retinal pigment cells (Kulkarni and Fingerman, 1987). We reported a neurotransmitter role for methionine-enkephalin in regulating the hemolymph sugar level of the freshwater crab Oziotelphusa senex senex, and hypothesized that methionine-enkephalin produces hyperglycemia indirectly by stimulating release of hyperglycemic hormone (Reddy, 1999).

The objectives of the present study were threefold: (a) by extending our studies to the estuarine crab Scylla serrata, to test our hypothesis, generated by the study of Oziotelphusa senex senex, that methionine-enkephalin produces hyperglycemia in decapod crustaceans; (b) to determine the changes in levels of tissue carbohydrates and phosphorylase activity during methionine-enkephalin treatment; and (c) to provide evidence that supports the triggering of release of hyperglycemic hormone during methionine-enkephalin treatment.

Materials and Methods

Individuals of Scylla serrata (15 [+ or -] 2 cm in carapace width; 110 [+ or -] 5 g wet weight) were collected from the Chennai coast, India. They were kept in large aquaria with continuous aeration and acclimatized to laboratory conditions for one week under constant salinity (25 [+ or -] 1 ppt), pH (7.2 [+ or -] 0.1), and temperature (23 [+ or -] 2[degrees]C). During this period the crabs were fed fish flesh. Feeding was stopped 24 h before the beginning of the experiments, and no food was given during experimentation. Only intermolt (Stage [C.sub.4]), intact, male crabs were used in the present study.

Methionine-enkephalin (Sigma Chemical Co.) was dissolved in physiological saline (Pantin, 1934). In these experiments, each of the 10 groups of crabs used consisted of 10 individuals. The first group served as normal and received no treatment. A second group served as control, with each crab in this group receiving an injection of 10 [micro]l of physiological saline (Pantin, 1934) through the base of the coxa of the 3rd pair of the walking legs. In groups 3-5 respectively, each crab received an injection of [10.sup.-7], and [10.sup.-8], and [10.sup.-9] mole methionine-enkephalin in 10 [micro]l volume. Both eyestalks ablated from all the crabs in groups 6-10. The eyestalks were extirpated by cutting them off at the base, without prior ligation but with cautery of the wound after operation. Twenty-four hours after eyestalk ablation, these groups were used for experimentation. Crabs in group 6 served simply as eyestalkless animals, and crabs in group 7 received 10 [micro]l crustacean Ringer solution and served a s eyestalkless controls. In groups 8-10 respectively, each crab was injected with [10.sup.-7], [10.sup.-8], and [10.sup.-9] mole methionine-enkephalin in 10 [micro]l volume. Based on preliminary kinetic studies, the crabs were sacrificed for analysis 2 h after injection (Figs. 1, 2).

Hemolymph (500 [micro]l) was aspirated by syringe, through the arthrodial membrane of the coxa of the 4th pair of walking legs. The other tissues (hepatopancreas and muscle from chela propodus) were then quickly dissected out, weighed, and analyzed by the procedures outlined below.

Hemolymph total carbohydrate level. Hemolymph total carbohydrate (TCHO) levels were estimated in trichloroacetic acid supernatant (10% TCA w/v) according to the method of Carroll et al. (1956).

Hemolymph glucose level. For measurement of glucose, 100 [micro]l of hemolymph was mixed with 300 [micro]l of 95% ethanol. After deproteinization (4 [degrees]C, 14,000 X g, 10 min), the sample was combined with a mixture of glucose enzyme reagent (glucose-6-phosphate dehydrogenase and NADP) and color reagents (phenazine methosulfate and iodonitrotetrazolium chloride) (kit from Sigma). After 30 min, the intensity of the color was measured at 490 nm and quantified with standards.

Tissue TCHO and glycogen levels. TCHO levels in the tissues (hepatopancreas and muscle) were estimated in the 10% TCA supernatant (5% w/v), and glycogen was estimated in the ethanolic precipitate of TCA supernatant, according to the method of Carroll et al. (1956).

To 0.5 ml of clear supernatant was added 5.0 ml of anthrone reagent, and the combination was boiled for 10 min in a water bath. The samples were then immediately cooled. A standard sample containing a known quantity of glucose solution was always tested along with the experimental samples. Absorbance was measured at 620 nm against a reagent blank.

Tissue phosphorylase activity. Phosphorylase activity was assayed in hepatopancreas and muscle by colorimetric determination of inorganic phosphate released from glucose-1-phosphate by the method of Cori et al. (1955). First, 0.4 ml of the enzyme was incubated with 2.0 mg of glycogen for 20 min at 35 [degrees]C, then the reaction was initiated by the addition of 0.2 ml of 0.016 M glucose-1-phosphate (G-1-P) to one tube (phosphorylase a) and a mixture of 0.2 ml of G-1-P and 0.004 M adenosine-5-monophosphate (phosphorylase ab) to another tube.

The reaction was incubated for 15 min for determining total phosphorylase and for 30 min for active phosphorylase. The reaction was terminated by the addition of 5.0 ml of 5 N sulfuric acid. Released inorganic phosphate was estimated by the method of Taussky and Shorr (1953).

Protein determination. Total protein levels in the enzyme source were estimated following the method of Lowry et al. (1951) using bovine serum albumin as standard.

Statistical analysis. Statistical analysis of the results was made using a two-way ANOVA test followed by Dunnet's multiple range test (preceded by one-way ANOVA), using SPSS version 10.0 (SPSS Inc., Chicago, IL).

Results

Effects of methionine-enkephalin on carbohydrate metabolism of intact crabs

Injection of methionine-enkephalin into intact crabs resulted in significant hyperglycemia and hypergiucosemia in a dose-dependent manner (Table 1), whereas injection of physiological saline had no effect on hemolymph carbohydrate levels. At doses between [10.sup.-9] mol/crab (36.41%) and [10.sup.-6] mol/crab (147.81%), the effect of methioine-enkephalin was statistically significant. For doses lower than [10.sup.-9] mol/crab, however, methionine-enkephalin did not elicit a hyperglycemic response (Fig. 1). A time course for methionine-enkephalin-induced hyperglycemia is shown in Figure 2 for a [10.sup.-7] mol/crab dose, which is a nearly saturating dose. The hemolymph glucose level increased significantly within 30 mm of methionine-enkephalin injection, reached a peak at 2 h, then declined gradually.

Hepatopancreas glycogen and TCHO levels in crabs that received methionine-enkephalin were significantly lower than those of control crabs (Table 2). Decreases in muscle glycogen and TCHO levels were also significant after the injection of methionine-enkephalin (Table 3), suggesting the possible mobilization of glucose molecules from hepatopancreas and muscle to hemolymph.

Phosphorylase (both total and active) activity levels were significantly increased in both hepatopancreas and muscle after the injection of methionine-enkephalin (Tables 4, 5). The ratio of active to total phosphorylase also increased in the tissues of crabs after the injection of methionine-enkephalin, indicating conversion of inactive to active phosphorylase.

Effects of bilateral eyestalk ablation and injection of methionine-enkephalin into ablated crabs on carbohydrate metabolism

Bilateral eyestalk removal caused a significant decrease in hemolymph carbohydrate level (Table 1). Enhancement of TCHO level of hepatopancreas and muscle was also significant in eyestalk-ablated crabs (Tables 2, 3). The increase was greater in muscle. Glycogen level in hepatopancreas increased significantly in eyestalkless crabs. A similar pattern was observed in muscle. Tissue phosphorylase activity levels decreased significantly in eyestalk-ablated crabs (Tables 4, 5).

Injection of methionine-enkephalin into eyestalkless crabs did not significantly change hemolymph carbohydrate levels compared to Ringer-injected eyestalkless crabs (Table 1). The levels of tissue TCHO and glycogen and activity levels of total and active phosphorylase were also not significantly altered in eyestalkless crabs after methionine-enkephalin injection (Tables 2-5).

Discussion

The effect of eyestalk hormones on tissue carbohydrate levels and phosphorylase activity has been extensively studied (Keller, 1965; Ramamurthi et al., 1968; Sagardia, 1969). Eyestalk removal inactivates the phosphorylase system and activates uridine-diphosphate-glucose glycogen transglucosylase (glycogen synthetase) (Keller, 1965; Ramamurthi et al., 1968). Ramamurthi et al. (1968) also observed stimulation of uptake and incorporation of glucose [blank.sup.14]C into the glycogen fraction of muscle tissue after eyestalk removal; this stimulation was accompanied by a decrease in hemolymph sugar level. Injection of eyestalk extract reversed these changes. The hyperglycemic hormone of eyestalks of the crab Oziotelphusa senex senex and the prawn Penaeus monodon enhances the activity of the phosphorylase system (Reddy et al., 1982, 1984; Reddy, 1992).

An increase in phosphorylase activity and a decrease in glycogen and TCHO levels in hepatopancreas and muscle of Scylla serrata, followed by hyperglycemia after the injection of methionine-enkephalin, indicate glycogenolysis and mobilization of sugar molecules from tissues to hemolymph. This is in agreement with other findings (see review by Reddy and Ramamurthi, 1999). Though the hormone that elevates hemolymph sugar is conventionally called crustacean hyperglycemic hormone (CHH), Hohnke and Scheer (1970) suggested that the primary function of the CHH is not to elevate hemolymph sugar level, but to elevate intracellular glucose through the degradation of glycogen by activating the enzyme phosphorylase. The conversion of phosphorylase from its inactive to active form results in glycogenolysis, and the resultant glucose molecules leak into the hemolymph, causing hyperglycemia. This view has been supported by Telford (1975).

Our results clearly demonstrate that methionine-enkephalin is involved in the regulation of carbohydrate metabolism in the crab Scylla serrata. In the present study, we show that methionine-enkephalin elicited a hyperglycemic response in S. serrata in a dose-dependent manner (Fig. 1). Methionine-enkephalin-induced hyperglycemia has been similarly demonstrated in the freshwater crab Oziotelphusa senex senex (Reddy, 1999) and the brackish-water prawns Penaeus indicus and Metapenaeus monocerus (Kishori et al., 2001). The doses of methionine-enkephalin that induced hyperglycemia ranged from [10.sup.-9] to [10.sup.-6] mol/animal (Fig. 1), which is comparable to those reported for O. senex senex (Reddy, 1999). Our observation that methionine-enkephalin was ineffective in inducing hyperglycemia in eyestalk-ablated S. serrata (Table 1) is also consistent with those obtained in crabs (Reddy, 1999) and prawns (Kishori et al., 2001) and suggests that the hyperglycemic effect of methionine-enkephalin results from an enh anced release of CHH (Keller, 1992; Soyez, 1997).

Injection of methionine-enkephalin into intact S. serrata also has two other effects. It activates the phosphorylase system, which causes degradation of glycogen. It also results in accumulation of sugar molecules in the tissues; these molecules are ultimately mobilized to hemolymph, causing hyperglycemia. Methionine-enkephalin might have elevated the phosphorylase system in intact crabs in several different ways--for example, by triggering release of hyperglycemic hormone or by mimicking the action of this hormone. However, because methionine-enkephalin was not able to produce these changes in eyestalkless crabs, it seems most likely that methionine-enkephalin exerted its hyperglycemic effect by triggering release of hyperglycemic hormone from the sinus gland of eyestalks. This supports our earlier results that sinus glands in the eyestalks of crabs are the main release site for hyperglycemic hormone (Reddy and Ramamurthi, 1982).

The mechanisms whereby methionine-enkephalin causes release of neurohormones are still uncertain. In mammals, endogenous opioid peptides are involved in regulating the release of neurohypophysial peptides (Bicknell et at., 1988; Yamada et at., 1988; Sasaki et at., 2000). In crustaceans, opioid-peptide-like (methionine-enkephalin-like, leucine-enkephalin-like and b-endorphin-like) hormones were isolated and characterized from X-organ sinus gland complexes of eyestalks (Fingerman et at., 1983, 1985). However, there is little information about the effect of opioid peptides on release of neurohormones in crustaceans. Sarojini et at. (1995, 1996), using highly selective opioid antagonists, provided evidence that methionine-enkephalin exerts its effect by acting through delta-type opioid receptors in regulating ovarian maturation in Procambarus clarkii. In vivo studies with tissues of P. clarkii showed that methionine-enkephalin exerted its effect by at least modulating the release of eyestalk peptide hormone (Sar ojini et at., 1997). Recently, we provided evidence for a neurotransmitter role for methionine-enkephalin in causing hyperglycemia in the crab 0. senex senex (Reddy, 1999). Methionine-enkephalin also triggers the release of red-pigmentconcentrating hormone, black-pigment-dispersing hormone (Quackenbush and Fingerman, 1984), and dark-pigmentadapting hormone (Kulkarni and Fingerman, 1987). Three facts make it seem likely that this hyperglycemic action of emthionine-enkephalin in the present study on S. serrata is also indirect and involves stimulation of release of CHH. Methionine-enkephalin-like material is present in the neuroendocrine complex of the eyestalk of crustaceans (Fingerman et at., 1983, 1985). Methionine-enkephalin mediation of release of neurohormones has been demonstrated (Reddy, 1999). In cases where methionine-enkephalin has been found to stimulate neurohormone release, it does not act in the absence of neuroendocrine organs. As further support for the conclusion, eyestalk extract from methion ine-enkephalin injected prawns showed significantly less activity than the normal eyestalk extract in inducing hyperglycemia (Kishori et at., 2001).

Although the mechanisms that trigger release of CHH are still unknown, it is noteworthy that 5-hydroxytryptamine (5-HT), or serotonin, triggers CHH release in the crayfish Orconectes limosus (Keller and Bayer, 1968), Astacus leptodactylus (Strolenberg and Van Herp, 1977), and Procambarus clarkil (Lee et al., 2000). Strolenberg and Van Herp (1977), working with A. leptodactylus, and Martin (1978), working with Porcellic dilatatus, found that the sinus glands of specimens injected with 5-HT show increased numbers of exocytotic profiles, suggestive of increased CHH release. Exocytosis in A. leptodactylus was maximal 2 h after 5-HT was injected, and the hemolymph glucose concentration peaked 4 h after the injection (Strolenberg and Van Herp, 1977). In P. dilatatus, hyperglycemia induced by 5-HT is mediated by 5-[HT.sub.1]- and 5-[HT.sub.2]-like receptors in triggering release of CHH (Lee et aL, 2000).

In summary, we have shown that methionine-enkephalin is a potent hyperglycemic regulator in the crab Scylla serrata. The most likely site of action of methionine-enkephalin is the eyestalks, where the X-organ-sinus glands may respond to methionine-enkephalin stimulation by releasing CHH. Based on these results, experiments are being conducted to determine whether methionine-enkephalin enhances the release of CHH in crustaceans.

Acknowledgments

We thank Prof. Armugam, University of Madras, Chennai, for supplying Scylla serrata and providing necessary laboratory facilities, and Dr. K. V. S. Sharma, Professor, Department of Statistics, Sri Venkateswara University, for analyzing the data. We also thank the anonymous reviewers whose comments improved our manuscript. We are grateful to Prof. R. Ramamurthi, Department of Zoology, for his encouragement. Mr. S. Umasankar and Miss B. Prema Sheela provided skilled technical assistance. This work was carried out with the financial assistance from Department of Science and Technology research grant (SP/SO/C04/96) to Dr. PSR. We also thank the staff, Department of Biotechnology, Sri Venkateswara University, for their invaluable assistance.

(*) To whom correspondence should be addressed. E-mail: psreddy@vsnl.com

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Table 1
Effect of eyestalk ablation (ESX) (24-h post-ablation) and injection of
methionine-enkephalin into intact and ablated crabs on hemolymph total
sugar and glucose levels in Scylla serrata
                                               No treatment
Total Sugar (mg/100 ml)
Intact                                      12.11 [+ or -] 1.01
(Group 1)
ESX                                         9.41 [+ or -] 1.13 b
(Group 2)                                        (-22.22)
Two-way ANOVA: [F.sub.1,90] (Between
groups)) = 772.002, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
98.747, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 94.552,
P [less than] 0.001.
Glucose (mg/100 mol)
Intact                                      6.41 [+ or -] 0.62
(Group 1)
ESX                                         5.19 [+ or -] 1.01 b
(Group 2)                                        (-19.03)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 440.810, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
40.092, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 44.753,
P[less than]0.001.
                                           Ringer injection
Total Sugar (mg/100 ml)
Intact                                    12.73 [+ or -] 1.84 a
(Group 1)                                       (5.12)
ESX                                       9.34 [+ or -] 1.03 b,c
(Group 2)                                       (-0.74)
Two-way ANOVA: [F.sub.1,90] (Between
groups)) = 772.002, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
98.747, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 94.552,
P [ less than] 0.001.
Glucose (mg/100 mol)
Intact                                    6.55 [+ or -] 0.76 a
(Group 1)                                       (2.16)
ESX                                       5.52 [+ or -] 0.81 b,c
(Group 2)                                       (6.35)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 440.810, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
40.092, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 44.753,
P[less than]0.001.
                                          [10.sup.-7] mol/crab
Total Sugar (mg/100 ml)
Intact                                     28.8 [+ or -] 2.18 b
(Group 1)                                       (126.23)
ESX                                        9.41 [+ or -] 1.13 b,c
(Group 2)                                        (0.74)
Two-way ANOVA: [F.sub.1,90] (Between
groups)) = 772.002, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
98.747, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 94.552,
P [less than] 0.001.
Glucose (mg/100 mol)
Intact                                    12.07b [+ or -] 1.34 b
(Group 1)                                       (84.27)
ESX                                        5.19 [+ or -] 1.01 b,c
(Group 2)                                       (-5.97)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 440.810, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
40.092, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 44.753,
P[less than]0.001.
                                          [10.sup.-8] mol/crab
Total Sugar (mg/100 ml)
Intact                                    19.64 [+ or -] 1.41 b
(Group 1)                                       (54.28)
ESX                                        9.43 [+ or -] 1.01 b,c
(Group 2)                                        (0.96)
Two-way ANOVA: [F.sub.1,90] (Between
groups)) = 772.002, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
98.747, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 94.552,
P [less than] 0.001.
Glucose (mg/100 mol)
Intact                                    11.44 [+ or -] 1.28 b
(Group 1)                                       (74.65)
ESX                                        5.21 [+ or -] 0.91 b,c
(Group 2)                                       (-5.61)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 440.810, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
40.092, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 44.753,
P[less than]0.001.
                                          [10.sup.-9] mol/crab
Total Sugar (mg/100 ml)
Intact                                    16.52 [+ or -] 1.94 b
(Group 1)                                       (29.77)
ESX                                        9.21 [+ or -] 1.08 b,c
(Group 2)                                       (-1.39)
Two-way ANOVA: [F.sub.1,90] (Between
groups)) = 772.002, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
98.747, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 94.552,
P [less than] 0.001.
Glucose (mg/100 mol)
Intact                                     9.13 [+ or -] 0.78 b
(Group 1)                                       (39.38)
ESX                                        5.44 [+ or -] 0.77 b,c
(Group 2)                                       (-1.44)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 440.810, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
40.092, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 44.753,
P[less than]0.001.
                                          Dunnet's comparison test
Total Sugar (mg/100 ml)
Intact                                    [F.sub.(4,45)] = 137.160
(Group 1)
ESX                                        [F.sub.(4,45)] = 0.099
(Group 2)
Two-way ANOVA: [F.sub.1,90] (Between
groups)) = 772.002, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
98.747, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 94.552,
P [less than] 0.001.
Glucose (mg/100 mol)
Intact                                    [F.sub.(4,45)] = 75.613
(Group 1)
ESX                                        [F.sub.(4,45)] = 0.387
(Group 2)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 440.810, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
40.092, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 44.753,
P[less than]0.001.
Values are mean [+ or -] SD of 10 individual crabs. Values in
parentheses are percent change from control. For calculation of
percent change for eyestalk-ablated (ESX) crabs and Ringer-injected
intact crabs, intact crabs served as control; for met-injected crabs,
Ringer-injected crabs served as control.
(a) Not significant compared with intact crabs.
(b) P [less than] 0.001 compared to intact crabs.
(c) Not significant compared to eyestalkless crabs.
Table 2
Effect of eyestalk ablation (ESX) (24-h post-ablation) and injection of
methionine-enkephalin into intact and ablated crabs on hepatopancreas
total carbohydrate (TCHO) and glycogen levels in Scylla serrata
                                               No treatment
TCHO (mg/g)
Intact                                      13.66 [+ or -] 1.54
(Group 1)
ESX                                        17.87 [+ or -] 1.94 b
(Group 2)                                         (30.96)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 566.317,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 19.027,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 14.896,
P [less than] 0.001.
Glycogen (mg/g)
Intact                                      1.22 [+ or -] 0.10
(Group 1)
ESX                                        2.04 [+ or -] 0.29 b
(Group 2)                                         (67.21)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 1658.593,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 24.964,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 27.016,
P [less than] 0.001.
                                              Ringer injection
TCHO (mg/g)
Intact                                      13.84 [+ or -] 1.61 a
(Group 1)                                          (1.32)
ESX                                        18.01 [+ or -] 1.97 b,c
(Group 2)                                          (0.67)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 566.317,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 19.027,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 14.896,
P [less than] 0.001.
Glycogen (mg/g)
Intact                                      1.23 [+ or -] 0.09 a
(Group 1)                                          (0.82)
ESX                                        2.06 [+ or -] 0.31 b,c
(Group 2)                                          (0.98)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 1658.593,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 24.964,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 27.016,
P [less than] 0.001.
                                            [10.sup.-7] mol/crab
TCHO (mg/g)
Intact                                      8.47 [+ or -] 0.97 b
(Group 1)                                         (-38.80)
ESX                                        17.44 [+ or -] 1.43 b,c
(Group 2)                                          (-0.74)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 566.317,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 19.027,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 14.896,
P [less than] 0.001.
Glycogen (mg/g)
Intact                                      0.58 [+ or -] 0.14 b
(Group 1)                                         (-52.84)
ESX                                        2.11 [+ or -] 1.18 b,c
(Group 2)                                          (2.42)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 1658.593,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 24.964,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 27.016,
P [less than] 0.001.
                                            [10.sup.-8] mol/crab
TCHO (mg/g)
Intact                                      9.01 [+ or -] 1.51 b
(Group 1)                                         (-34.89)
ESX                                        17.81 [+ or -] 1.62 b,c
(Group 2)                                          (-0.96)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 566.317,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 19.027,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 14.896,
P [less than] 0.001.
Glycogen (mg/g)
Intact                                      0.61 [+ or -] 0.13 b
(Group 1)                                         (-50.40)
ESX                                        2.09 [+ or -] 0.21 b,c
(Group 2)                                          (1.45)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 1658.593,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 24.964,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 27.016,
P [less than] 0.001.
                                            [10.sup.-9] mol/crab
TCHO (mg/g)
Intact                                      9.47 [+ or -] 1.49 b
(Group 1)                                         (-31.57)
ESX                                        17.93 [+ or -] 1.59 b,c
(Group 2)                                          (-1.39)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 566.317,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 19.027,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 14.896,
P [less than] 0.001.
Glycogen (mg/g)
Intact                                      0.64 [+ or -] 0.21 b
(Group 1)                                         (-47.96)
ESX                                        2.07 [+ or -] 0.28 b,c
(Group 2)                                          (0.48)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 1658.593,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 24.964,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 27.016,
P [less than] 0.001.
                                           Dunnet's comparison test
TCHO (mg/g)
Intact                                     [F.sub.(4,45)] = 38.033
(Group 1)                                    P [less than] 0.001
ESX                                         [F.sub.(4,45)] = 0.229
(Group 2)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 566.317,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 19.027,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 14.896,
P [less than] 0.001.
Glycogen (mg/g)
Intact                                     [F.sub.(4,45)] = 148.477
(Group 1)                                    P [less than] 0.001
ESX                                         [F.sub.(4,45)] = 0.230
(Group 2)
Two-way ANOVA: [F.sub.1,90]
(Between groups) = 1658.593,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 24.964,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 27.016,
P [less than] 0.001.
Values are mean [+ or -] SD of 10 individual crabs. Values in
parentheses are percent change from control. For calculation of percent
change for ESX crabs and Ringer-injected intact crabs, intact crabs
served as control; for met-injected crabs, Ringer-injected crabs served
as control.
(a) Not significant compared with intact crabs.
(b) P [less than] 0.001 compared to intact crabs.
(c) Not significant compared to eyestalkless crabs.
Table 3
Effect of eyestalk ablation (ESX) (24 h post-ablation) and injection of
methio-enkephalin into intact and ablatedcrabs on muscle total
carbohydrate (TCHO) and glycogen levels in Scylla serrata
                                              No treatment
TCHO (mg/g)
Intact                                     4.39 [+ or -] 0.53
(Group I)
ESX                                       6.26 [+ or -] 0.71 b
(Group 2)                                       (42.59)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 579.612,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 8.707,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 9.114,
P [less than] 0.001.
Glycogen (mg/s)
Intact                                     0.66 [+ or -] 0.06
(Group 1)
ESX                                       1.01 [+ or -] 0.09 b
(Group 2)                                       (53.03)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 422.031, P [less than] 0.001;
[F.sub.4,90] Among treatments) = 6.391,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 6.713,
P [less than] 0.001.
                                             Ringer injection
TCHO (mg/g)
Intact                                     4.41 [+ or -] 0.49 a
(Group I)                                         (0.46)
ESX                                       6.31 [+ or -] 0.81 b,c
(Group 2)                                         (0.80)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 579.612,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 8.707,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 9.114,
P [less than] 0.001.
Glycogen (mg/s)
Intact                                     0.64 [+ or -] 0.09 a
(Group 1)                                        (-3.03)
ESX                                       1.02 [+ or -] 0.11 b,c
(Group 2)                                         (0.99)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 422.031, P [less than] 0.001;
[F.sub.4,90] Among treatments) = 6.391,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 6.713,
P [less than] 0.001.
                                           [10.sup.7] mol/crab
TCHO (mg/g)
Intact                                     2.94 [+ or -] 0.31 b
(Group I)                                        (-33.33)
ESX                                       6.31 [+ or -] 0.76 b,c
(Group 2)                                          (0)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 579.612,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 8.707,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 9.114,
P [less than] 0.001.
Glycogen (mg/s)
Intact                                     0.34 [+ or -] 0.09 b
(Group 1)                                        (-46.87)
ESX                                       0.99 [+ or -] 0.14 b,c
(Group 2)                                        (-2.94)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 422.031, P [less than] 0.001;
[F.sub.4,90] Among treatments) = 6.391,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 6.713,
P [less than] 0.001.
                                               [10.sup.-8]
TCHO (mg/g)
Intact                                     3.01 [+ or -] 0.37 b
(Group I)                                        (-31.74)
ESX                                       6.25 [+ or -] 0.84 b,c
(Group 2)                                        (-0.95)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 579.612,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 8.707,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 9.114,
P [less than] 0.001.
Glycogen (mg/s)
Intact                                     0.37 [+ or -] 0.06 b
(Group 1)                                        (-42.18)
ESX                                       1.07 [+ or -] 0.33 b,c
(Group 2)                                         (4.90)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 422.031, P [less than] 0.001;
[F.sub.4,90] Among treatments) = 6.391,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 6.713,
P [less than] 0.001.
                                           [10.sup.-9] mol/crab
TCHO (mg/g)
Intact                                     3.12 [+ or -] 0.92 b
(Group I)                                        (-29.25)
ESX                                       6.33 [+ or -] 0.92 b,c
(Group 2)                                         (0.31)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 579.612,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 8.707,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 9.114,
P [less than] 0.001.
Glycogen (mg/s)
Intact                                     0.41 [+ or -] 0.08 b
(Group 1)                                        (-35.31)
ESX                                       1.03 [+ or -] 0.21 b,c
(Group 2)                                         (0.98)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 422.031, P [less than] 0.001;
[F.sub.4,90] Among treatments) = 6.391,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 6.713,
P [less than] 0.001.
                                          Dunnet's comparison treat
TCHO (mg/g)
Intact                                     [F.sub.(4,45)] = 30.829
(Group I)                                    P [less than] 0.001
ESX                                        [F.sub.(4,45)] = 0.045
(Group 2)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 579.612,
P [less than] 0.001;
[F.sub.4,90] (Among treatments) = 8.707,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 9.114,
P [less than] 0.001.
Glycogen (mg/s)
Intact                                     [F.sub.(4,45)] = 45.114
(Group 1)                                    P [less than] 0.001
ESX                                        [F.sub.(4,45)] = 0.188
(Group 2)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 422.031, P [less than] 0.001;
[F.sub.4,90] Among treatments) = 6.391,
P [less than] 0.001;
[F.sub.4,90] (Interaction) = 6.713,
P [less than] 0.001.
Values are mean (mg glucose/g tissue) [+ or -] SD of 10 individuals
crabs. Values in parentheses are percent change from control. For
calculation of percent change for ESX crabs and Ringer-injected intact
crabs, intact crabs served as control; for met-injected crabs, Ringer-
injected crabs served as control.
(a) Not significant compared with intact crabs.
(b) P [less than]0.001 compared to intact crabs.
(c) Not significant compared to eyestalkless crabs.
Table 4
Effect of eyestalk ablation (ESX) (24 h post-ablation) and injection
of methionine-enkephalin into intact and ablated crabs on
hepatopancreas phosphorylase activity levels in Scylla serrata
                                             No treatment
Phosphorylase a
Intact                                    2.62 [+ or -] 0.29
(Group 1)
ESX                                      1.72 [+ or -] 0.31 b
(Group 2)                                      (-34.35)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 716.848, p [less than]
0.001; [F.sub.4,90] (Among treatments)
= 23.852, p [less than] 0.001;
[F.sub.4,90] (Interaction) = 18.208,
P [less than] 0.001.
Phosporylase ab
Intact                                    4.52 [+ or -] 0.41
(Group 1)
ESX                                      4.06 [+ or -] 0.44 b
(Group 2)                                      (-10.18)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 169.103, P [less than] 0.001;
[F.sub.4,90] (Among treatments)
= 11.291, P [less than] 0.001;
[F.sub.4,90] (Interaction) 9.985,
P [less than] 0.001.
                                            Ringer injection
Phosphorylase a
Intact                                    2.67 [+ or -] 0.33 a
(Group 1)                                        (1.91)
ESX                                      1.67 [+ or -] 0.29 b,c
(Group 2)                                       (-2.33)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 716.848, p [less than]
0.001; [F.sub.4,90] (Among treatments)
= 23.852, p [less than] 0.001;
[F.sub.4,90] (Interaction) = 18.208,
P [less than] 0.001.
Phosporylase ab
Intact                                    4.56 [+ or -] 0.44 a
(Group 1)                                        (0.89)
ESX                                      4.08 [+ or -] 0.41 b,c
(Group 2)                                        (0.49)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 169.103, P [less than] 0.001;
[F.sub.4,90] (Among treatments)
= 11.291, P [less than] 0.001;
[F.sub.4,90] (Interaction) 9.985,
P [less than] 0.001.
                                          [10.sup.-7] mol/crab
Phosphorylase a
Intact                                    3.87 [+ or -] 0.46 b
(Group 1)                                       (44.94)
ESX                                      1.69 [+ or -] 0.11 b,c
(Group 2)                                        (1.19)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 716.848, p [less than]
0.001; [F.sub.4,90] (Among treatments)
= 23.852, p [less than] 0.001;
[F.sub.4,90] (Interaction) = 18.208,
P [less than] 0.001.
Phosporylase ab
Intact                                    5.81 [+ or -] 0.67 b
(Group 1)                                       (27.41)
ESX                                      4.10 [+ or -] 0.39 b,c
(Group 2)                                        (0.49)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 169.103, P [less than] 0.001;
[F.sub.4,90] (Among treatments)
= 11.291, P [less than] 0.001;
[F.sub.4,90] (Interaction) 9.985,
P [less than] 0.001.
                                          [10.sup.-8] mol/crab
Phosphorylase a
Intact                                    3.63 [+ or -] 0.34 b
(Group 1)                                       (35.95)
ESX                                      1.81 [+ or -] 0.22 b,c
(Group 2)                                        (8.38)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 716.848, p [less than]
0.001; [F.sub.4,90] (Among treatments)
= 23.852, p [less than] 0.001;
[F.sub.4,90] (Interaction) = 18.208,
P [less than] 0.001.
Phosporylase ab
Intact                                    5.69 [+ or -] 0.52 b
(Group 1)                                       (24.78)
ESX                                      4.12 [+ or -] 0.34 b,c
(Group 2)                                        (0.98)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 169.103, P [less than] 0.001;
[F.sub.4,90] (Among treatments)
= 11.291, P [less than] 0.001;
[F.sub.4,90] (Interaction) 9.985,
P [less than] 0.001.
                                          [10.sup.-9] mol/crab
Phosphorylase a
Intact                                    3.60 [+ or -] 0.42 b
(Group 1)                                       (34.83)
ESX                                      1.84 [+ or -] 0.31 b,c
(Group 2)                                       (10.17)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 716.848, p [less than]
0.001; [F.sub.4,90] (Among treatments)
= 23.852, p [less than] 0.001;
[F.sub.4,90] (Interaction) = 18.208,
P [less than] 0.001.
Phosporylase ab
Intact                                    5.56 [+ or -] 0.73 b
(Group 1)                                       (21.92)
ESX                                      4.09 [+ or -] 0.51 b,c
(Group 2)                                        (0.24)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 169.103, P [less than] 0.001;
[F.sub.4,90] (Among treatments)
= 11.291, P [less than] 0.001;
[F.sub.4,90] (Interaction) 9.985,
P [less than] 0.001.
                                         Dunnet's comparison test
Phosphorylase a
Intact                                    [F.sub.(4,45)] = 28.430
(Group 1)                                  P [less than] 0.0001
ESX                                       [F.sub.(4,45)] = 1,473
(Group 2)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 716.848, p [less than]
0.001; [F.sub.4,90] (Among treatments)
= 23.852, p [less than] 0.001;
[F.sub.4,90] (Interaction) = 18.208,
P [less than] 0.001.
Phosporylase ab
Intact                                    [F.sub.(4,45)]= 15.846
(Group 1)                                  P [less than] 0.001
ESX                                       [F.sub.(4,45)] = 0.044
(Group 2)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 169.103, P [less than] 0.001;
[F.sub.4,90] (Among treatments)
= 11.291, P [less than] 0.001;
[F.sub.4,90] (Interaction) 9.985,
P [less than] 0.001.
Value are mean (iP released/mg protein/h [+ or -] SD of 10 individual
crabs. Values in parentheses are percent change from control. For
calculation of percent change for ESX crabs and Ringer-injected intact
crabs, intact crabs served as control; for met-injected crabs, Ringer-
injected crabs served as control.
(a) Not significant compared with intact crabs.
(b) P [less than] 0.001 compared to intact crabs.
(c) Not significant compared to eyestalkless crabs.
Table 5
Effect of eyestalk ablation (ESX) (24 h post-ablation) and injection of
methinine-enkephalin into intact and ablated crabs on muscle
phosphorylase activity levels in Scylla serrata
                                             No treatment
Phosphorylase a
Intact                                    1.92 [+ or -] 0.09
(Group 1)
ESX                                       0.99 [+ or -] 0.08 b
(Group 2)                                      (-48.44)
Two-way ANOVA: [F.sub.1,90] Between
groups) = 1711.188, P [less than]
0.001; [F.sub.4,90] (Among treatments) =
58.745, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 52,927,
P [less than] 0.001.
Phosphorylase ab
Intact                                    2.49 [+ or -] 0.45
(Group 1)
ESX                                       2.22 [+ or -] 0.32 b
(Group 2)                                      (-11.65)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 136.048, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
10.259, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 8.734,
P [less than] 0.001.
                                           Ringer injection
Phosphorylase a
Intact                                    1.94 [+ or -] 0.14 a
(Group 1)                                       (1.04)
ESX                                       1.02 [+ or -] 0.11 b,c
(Group 2)                                       (3.03)
Two-way ANOVA: [F.sub.1,90] Between
groups) = 1711.188, P [less than]
0.001; [F.sub.4,90] (Among treatments) =
58.745, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 52,927,
P [less than] 0.001.
Phosphorylase ab
Intact                                    2.52 [+ or -] 0.49 a
(Group 1)                                       (1.21)
ESX                                       2.18 [+ or -] 0.31 b,c
(Group 2)                                      (-0.91)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 136.048, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
10.259, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 8.734,
P [less than] 0.001.
                                          [10.sup.-7] mol/crab
Phosphorylase a
Intact                                     3.26 [+ or -] 0.36 b
(Group 1)                                       (68.04)
ESX                                        1.01 [+ or -] 0.09 b,c
(Group 2)                                       (-0.74)
Two-way ANOVA: [F.sub.1,90] Between
groups) = 1711.188, P [less than]
0.001; [F.sub.4,90] (Among treatments) =
58.745, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 52,927,
P [less than] 0.001.
Phosphorylase ab
Intact                                     3.49 [+ or -] 0.41 b
(Group 1)                                       (38.49)
ESX                                        2.22 [+ or -] 0.34 b,c
(Group 2)                                        (1.83)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 136.048, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
10.259, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 8.734,
P [less than] 0.001.
                                          [10.sup.-8] mol/crab
Phosphorylase a
Intact                                     3.01 [+ or -] 0.12 b
(Group 1)                                       (55.15)
ESX                                        1.04 [+ or -] 0.13 b,c
(Group 2)                                        (1.96)
Two-way ANOVA: [F.sub.1,90] Between
groups) = 1711.188, P [less than]
0.001; [F.sub.4,90] (Among treatments) =
58.745, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 52,927,
P [less than] 0.001.
Phosphorylase ab
Intact                                     3.46 [+ or -] 0.44 b
(Group 1)                                       (37.30)
ESX                                        2.24 [+ or -] 0.42 b,c
(Group 2)                                        (2.75)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 136.048, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
10.259, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 8.734,
P [less than] 0.001.
                                          [10.sup.-9] mol/crab
Phosphorylase a
Intact                                     3.02 [+ or -] 0.26 b
(Group 1)                                       (55.67)
ESX                                        1.06 [+ or -] 0.21 b,c
(Group 2)                                        (3.92)
Two-way ANOVA: [F.sub.1,90] Between
groups) = 1711.188, P [less than]
0.001; [F.sub.4,90] (Among treatments) =
58.745, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 52,927,
P [less than] 0.001.
Phosphorylase ab
Intact                                     3.44 [+ or -] 0.51 b
(Group 1)                                       (36.50)
ESX                                        2.25 [+ or -] 0.41 b,c
(Group 2)                                        (3.21)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 136.048, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
10.259, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 8.734,
P [less than] 0.001.
                                          Dunnet's comparison test
Phosphorylase a
Intact                                    [F.sub.(4,45)] = 84.853
(Group 1)                                   P [less than] 0.001
ESX                                       [F.sub.(4,45)] = 0.368
(Group 2)
Two-way ANOVA: [F.sub.1,90] Between
groups) = 1711.188, P [less than]
0.001; [F.sub.4,90] (Among treatments) =
58.745, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 52,927,
P [less than] 0.001.
Phosphorylase ab
Intact                                    [F.sub.(4,45)] = 16.086
(Group 1)                                   P [less than] 0.001
ESX                                        [F.sub.(4,45)] = 0.061
(Group 2)
Two-way ANOVA: [F.sub.1,90] (Between
groups) = 136.048, P [less than] 0.001;
[F.sub.4,90] (Among treatments) =
10.259, P [less than] 0.001;
[F.sub.4,90] (Interaction) = 8.734,
P [less than] 0.001.
Values are mean (iP released/mg protein/h) [+ or -] SD of 10 individual
crabs. Values in parentheses are percent change from control. For
calculation of percent change for ESX crabs and Ringer-injected crabs,
intact crabs served as control; for met-injected crbas, Ringer-injected
crabs served as control.
(a) Not siginificant compared with intact crabs.
(b) P [less than] 0.001 compared to intact crabs.
(c) Not significant compared to eyestalkless crabs.
Figure 1.
Dose-dependent effect of methionine-enkephalin on the hemolymph glucose
levels in intact Scylla serrata. Two hours after injection of saline
(10 [micro]l/animal) or methionine-enkephalin at the doses indicated,
hemolymph was withdrawn from crabs for glucose determination. Each bar
represents a mean [+ or -] SD (n = 10). Numbers in parentheses
indicates the percent increase from the normal values.
[Methionine - Enkephatin] (mol/crab)  Hemolymph glucose (mg/100ml)
NORMAL
SALINE-INJECTED                                  (5.11) NS
[10.sup.-10]                                     (4.54) NS
[10.sup.-9]                                     (36.41) *
[10.sup.-8]                                     (62.18) *
[10.sup.-7]                                    (137.81) *
[10.sup.-6]                                    (147.81) *
[10.sup.-5]                                    (145.08) *
(*)Significant difference from normal crabs at P [less than] 0.001.
(NS) Not significant.
Note: Table made from bar graph


[Graph omitted]
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Author:REDDY, P. SREENIVASULA; KISHORI, B.
Publication:The Biological Bulletin
Geographic Code:1USA
Date:Aug 1, 2001
Words:8745
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