A neurotransmitter role for methionine enkephalin in causing hyperglycemia in the freshwater crab, Oziotelphusa senex senex

P. Sreenivasula Reddy

Department of Biotechnology, S. V. University, Tirupati 517 512, India

Injection of methionine enkephalin caused a significant increase in the hemolymph glucose and total sugar level of intact crabs in a dose-dependent manner, apparently by triggering release of the hyperglycemic hormone.

EVER since the first report of presence of leucine enkephalin in the red swamp crayfish, Procambarus clarkii and in the spiny lobster, Panulirus interruptus by Mancillas et al.1, there have been sporadic reports of occurrence of opioids in different crustacean species2–6. Though the status of opioid research in crustaceans is fragmentary, there are several interesting questions pertaining to demonstration of these peptides and the diverse functions they perform, which are very different from what they do in mammals7,8.

Since the discovery of a ‘diabetogenic factor’ in the crustacean eyestalk9, work has been carried out on its chemical nature, mode and site of action10–14. In view of the fact that opioid peptides act as neurotransmitters6,15, it is conceivable that these peptides could help in the secretion of the hyperglycemic hormone from the neurosecretory cells that synthesize it. The present investigation was undertaken to determine, whether methionine enkephalin can indeed produce an increase in the hemolymph sugar level of the crab Oziotelphusa senex senex and if so whether it might involve stimulation of the release of hyperglycemic hormone.

Freshwater rice field crabs Oziotelphusa (Paratelphusa) senex senex Fabricius were collected

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from local paddy fields in and around Tirupati and maintained at room temperature 27 ±  1° C in plastic tubs half submerged in tap water with a 12L:12D cycle. They were fed daily once ad libitum sheep meat and the medium was changed. Only adult, healthy, intermolt (stage C4) male crabs with a carapace width of 30–32 mm and a body weight of 32–34 g were used in the present study.

One hundred crabs were divided into 10 groups of 10 animals each. Group 1 served as normal and this group of crabs which received no treatment were utilized on the first day of the experiment. Group 2 received 20 ml of crustacean saline through the arthrodial membrane of the coxa of the 3rd pair of walking legs and served as controls. Groups 3, 4 and 5 received 10–8, 10–9 and 10–10 mol/crab of methionine enkephalin, respectively.

Eyestalks were removed from the remaining groups (groups 6–10) by cutting off the organs at the base without prior ligation but with cautery of wound after operation. After 24 h of eyestalk ablation, these groups were analysed for hemolymph sugar level. Crabs from group 6 were sacrificed without treatment while those from group 7 received only 20 ml crustacean saline. Groups 8–10 received 10–8, 10–9 and 10–10 mol/crab of methionine enkephalin.

Methionine enkephalin purchased from the Sigma Chemical Company, St. Louis, Missouri, was dissolved in physiological saline and injected in 20 ml of volume. All hemolymph samples were removed two hours after injection and at the same time of the day to obviate any possible variation due to circadian rhythm in the hemolymph sugar level. The hemolymph sugar level was determined using anthrone reagent16. For the measurement of glucose, hemolymph (100 m l) was mixed with 300 m l of 95% ethanol. After deproteinization (4° C, 14,000 g, 10 min), the sample was mixed with a mixture of glucose enzyme reagent (glucose-6-phosphate dehydrogenase and NADP) and colour reagents (phenazine methosulfate and iodonitrotetrazolium chloride) (kit from Sigma). After 30 min, the intensity of the colour was measured at 490 nm and quantified with standards. One way ANOVA test was employed to analyse the data followed by Duncan’s multiple range test to determine the level of significance.

The hemolymph total reducing sugar level in eyestalk-less crabs was significantly (P < 0.001) less (–25.98%) than in intact crabs. Methionine enkephalin significantly (P < 0.001) increased the hemolymph sugar level in intact crabs but not in eyestalk-less crabs (Table 1). These results revealed that not only did methionine enkephalin produce hyperglycemia in intact crabs, but also that this hyperglycemia was dose-dependent. Eyestalk ablation also resulted in hypoglucosemia (Table 1). Injection of methionine enkephalin elevated hemolymph glucose level in a dose-dependent manner in intact crabs. Administration of methionine enkephalin did not cause any

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change in hemolymph glucose level in eyestalk ablated crabs. Injection of physiological saline did not yield any significant change in hemolymph glucose and sugar level in intact as well as eyestalk-less crabs.

The data indicate that eyestalk removal can lower both hemolymph sugar and glucose concentrations. Eyestalk extirpation is a classical operation of crustacean endocrinology; it removes the X-organ-sinus gland complex which is the source of an array of hormones17, including the hyperglycemic hormone. Removal of eyestalks eliminates hyperglycemic hormone from circulation which results in significant decrease in hemolymph sugar level. We have reported earlier that hyperglycemic hormone is synthesized in and released from the sinus glands of eyestalks of the crab, Oziotelphusa senex senex18.

Methionine enkephalin could have produced a rise in hemolymph sugar level in the intact crabs in several different ways such as by triggering release of hyperglycemic hormone, by mimicking the action of this hormone or even by directly stimulating glycogenolysis. However, because methionine enkephalin was not able to produce an increase in hemolymph sugar level in eyestalkless crabs, it seems most likely that methionine enkephalin exerted its hyperglycemic effect by triggering the release of hyperglycemic hormone from the sinus gland of the eyestalks. This also supports our earlier results18 that the sinus glands in the eyestalks of this crab are the main release sites of hyperglycemic hormone. These results provide the first evidence that an opioid peptide is involved in the regulation of hemolymph sugar level in a crustacean and supports the hypothesis that methionine enkephalin has a neurotransmitter role in decapod crustaceans19,20.

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ACKNOWLEDGEMENTS.  I thank the Management, Pondicherry University, Pondicherry for granting sabbatical leave during which the present work was carried out. I also thank the Staff, Department of Zoology, S.V. University for their invaluable assistance. Portions of this work are a result of research supported by DST (SP/SO/CO4/96), New Delhi. Dr K.V.S. Sarma, Associate Professor, Department of Statistics helped in the analysis of the data.

Received 22 July 1998; revised accepted 9 February 1999