Moult-modulating effect of serotonin in the crab Oziotelphusa senex senex (Fabricius).
Arthropod invertebrates such as insects and crustaceans require periodic moulting to attain somatic growth. Further among crustaceans the cycles of growth and moulting are controlled by steroid hormones such as ecdysteroids secreted by paired endocrine glands the Yorgan (Lachaise et al. 1993; Webster 1998). The sinus gland complex present in the eyestalks is the major endocrine center in crustaceans. Moult inhibiting hormone (MIH), one of the important neurohormones synthesized and released from the X-organ sinus gland complex is primarily involved in the regulation of moulting (Lachaise et al. 1993). Within this group apart from the peptides that inhibit moulting, are those that have been functionally identified as crustacean hyperglycemic hormone (CHH) and mandibular organ inhibiting hormone (MOIH) (Webster 1998). In crabs MIH and CHH are active in repressing ecdysteroid synthesis by Y-organ (Webster and Keller 1986). The fact that neuromodulators including neurotransmitters mediate and control a wide variety of physiological actions of neurohormones has been discussed elsewhere (Vaca and Alfaro 2000).
Biogenic amines have been found to modulate the release of various neurohormones from crustacean neuroendocrine tissue. 5-Hydroxytryptamine (5-HT) is an amine-derived neurotransmitter implicated in the regulation of a wide variety of physiological and behavioral processes of vertebrates and invertebrates (Zifa and Fillion 1992; Fingerman 1997). In vertebrates, 5-HT acts through a membrane-bound G-protein coupled receptor that stimulates the signal transduction pathway. Serotonin (5-HT) has been reported to stimulate the release of moult inhibiting hormone (MIH) (Mattson and Spaziani 1985), the crustacean hyperglycemic hormone (CHH) (Keller and Beyer 1968) the neurodepressing hormone (NDH) (Arechiga et al. 1985) and red pigment dispersing hormone (RPDH) (Rao and Fingerman 1970). Serotonin has also been implicated as a regulator of life-long neurogenesis in the lobster brain (Benton and Beltz 2001; Beltz et al. 2001). In view of the importance of aminergic neurotransmitters in the regulation of various crustacean neurohormones, an attempt has been made to elucidate the modulatory effect of serotonin on moulting in the Indian freshwater field crab Oziotelphusa senex senex (Fabricius).
MATERIALS AND METHODS
Adult, healthy crabs, O. senex senex procured from local paddy fields prior to the application of pesticides, were acclimated to laboratory conditions for about a week. They were kept in large plastic troughs containing filtered tap water (pH 7.3 [+ or -] 1; temperature 25[+ or -] 2C; oxygen 5-6 PPM). The crabs fed daily on fresh muscle pieces of frog and the water in the troughs was changed following feeding in order to remove remnants of food particles. Intermoult male crabs of similar body size (25 [+ or -] 2g) of C4 (Intermoult) stage (as determined by Drach (1939) and Stevenson (1972)) were selected for experimentation to avoid possible variations due to sex, size and moulting effects. The crabs were divided into four groups of 50 each. Crabs from all the groups were induced to moult by removing four pairs of walking legs (Skinner and Graham 1974) since the moulting activity in the intact crabs is generally low. Immediately after removing the walking legs Vaseline was applied at the limbs in order to avoid loss of body fluids. One of the groups served as control while others, which were given injections of serotonin @ 1x10-8, 1x10-7, and 1x[10.sup.-6] per crab respectively served as experimental groups. The injections were given on day 1, 11 and 21. Moulting was observed until each crab exhibited at least one moult within the experimental duration of 50 days.
No mortalities occurred in either experimental groups or in the control groups. The crabs those lacking four legs that served as controls that underwent ecdysis at a rapid rate. First Moulting was observed on 21st day of the experiment (Fig.1). However, the crabs received injections of serotonin underwent ecdysis at a much slower rate than did the controls (Fig.1). The degree of moult inhibition increased with dose of serotonin. Depending on the dose given, serotonin caused either a delay in the incipience of moult or a decrease in the percentage of moulting individuals. First Moulting was observed on 29th, 34th and 39th day of experiment for the serotonin doses @1x[10.sup.-8], 1x[10.sup.-7] and 1x[10.sup.-6] mol/crab respectively (table 1) . When compared with the controls, the percentage moult inhibition was 42%, 70% and 86% for serotonin doses @1x[10.sup.-8], 1x[10.sup.-]7 and 1x[10.sup.-6] mol/crab.
The crabs received injections of serotonin underwent ecdysis at a much slower rate than did the controls in a dose dependent mode. A possible explanation for a relationship between serotonin and incidence of moult delay is related to the neuroendocrine control of moulting. Concepts of hormonal control of moulting were in-fact first made several decades ago (Kopec 1922). The interrelationship between X-organ and Y-organ in crustacean moulting has been well documented (Skinner 1985; Lachaise et al. 1993). The inhibition of moulting process recruitment into premoult is mediated by steroid hormone, 20-hydroxy ecdysone. The secretory source of the steroid hormone is the Y-organ (Lachaise and Feyereisen 1976; Chang and O'Connor 1988; Vijayan et al. 1993). The rate of synthesis and secretion of ecdysone by the Y-organ is negatively regulated by the X-organ sinus gland complex. It is well known that the circulating titer of 20-hydroxyecdysone varies along the molt cycle. Immediately after ecdysis, the titer is low and generally remains so during intermolt. A major increase occurs at stage D1 -D2 followed by an abrupt drop just before the moulting (Chang, 1992). The serotonin may affect the X-organ in such a way that the production and release of moult inhibiting hormone continued beyond the normal time, the effect would be to delay in initiation of ecdysis. Another possible cause of delay is that serotonin act directly on Y-organs and affects the synthesis and release of ecdysone by the Y-organ. Ecdysteroids regulate gene activities at the transcriptional level through binding with the ecdysteroid receptor which ultimately heterodimerizes with ultraspiracle protein (Oberdorster and Cheek 2000). This dimer binds to specific DNA response elements in the genes that code for the enzymes responsible for exoskeleton degradation such as chitobiase that are regulated by the molting hormones (Zou 2005). It is necessary to note that chitobiase activity in general varied between tissues and the moult cycle stage with control most likely under the influence of the crab's steroid moulting hormones (Zou and Fingerman 1999). Inhibition of synthesis and release of ecdysone by the serotoin might not be facilitating the transcription of the genes that codes for the enzymes such as chitobiase.
Another possible cause of delay of moulting may be the crabs enter into reproduction and starts synthesizing vitellogenin. We observed that ovarian index, oocyte diameter and ovarian total lipid content increased in serotonin-injected crabs (data not shown). The sterols available through the diet are utilizing for synthesizing the vitellogenin and moulting thereby delayed. However, the exact mechanism of inhibition of moulting can not be speculated from this data.
The results of this investigation suggest that the moulting in crustaceans might be altered by the biogenic amines.
I thank Prof. K. V. Ramana Rao, for his help and advice, and the Department of Zoology, Sri Venkateswara University, Tirupati, for providing laboratory facilities.
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L. V. K. S. Bhaskar Department of Zoology, Sri Venkateswara University, Tirupati-517 502 (AP), India
Table 1. Observations regarding incipience of moulting (day), number of animals and percentage of animals moulted during the experimental period in control and experimental (1x[10.sup.s], 1x[10.sup.-7] and 1x[10.sup.-6] mol. serotonin injected) crab O. senex senex. Incipience of Number of animals Percentage of the moulting(day) moulted during the animals moulted experimental period during experimen- tal period Control 21 50 100 1x[.sup.10-8] 29 29 58 1x[10.sup.-7] 34 15 30 1x[10.sup.-6] 39 7 14