Endocrine disruption can be defined as the interaction of exogenous xenobiotics with the endocrine systems of organisms. The phenomenon is well documented in vertebrate species including man (Sharpe and Skakkebaek 1993, Colborn et al 1996). The effect of vertebrate type hormones on aquatic organisms has received considerable investigation, with reports of sex changes in riverine fish (Sumpter 1995) and marine snails (Matthiessen and Gibbs 1998) and abnormalities in the reproductive organs of alligators (Guillette et al 1994).
Less is known regarding the potential for chemicals to disrupt endocrine processes in invertebrate species (see Depledge and Billinghurst 1999 for review). Since invertebrates account for 95% of all described animal species and are vital components of marine ecosystems (deFur et al 1999), it is pertinent to study the potential effects of anthropogenic contaminants on their hormonally regulated functions. Investigations into endocrine disruption in invertebrates have thus far chiefly focussed on the potential influence of vertebrate type hormones and their analogues. There is little direct evidence that natural and anthropogenic compounds capable of modifying endocrine control in vertebrates have similar effects in invertebrates such as crustaceans (see Pinder et al 2000 for review). Investigations at the Plymouth Environmental Research Centre (PERC) carried out as part of the EDMAR programme did not show any alterations in the hormonally controlled processes of vitellogenesis, heart rate or osmoregulation in shore crabs (Carcinus maenas) following their exposure to various natural and synthetic vertebrate sex steroids. Despite the gaps in our knowledge regarding invertebrate endocrine sytems, preliminary findings suggest that the pathways and chemical messengers used by invertebrates differ from vertebrate species. Thus, exogenous sources of oestrogen and its mimics together with androgens do not appear to have any significant effects on the endocrine controlled physiological or reproductive processes investigated in this decapod species.
Little is known of the potential for exogenous contaminants to mimic or antagonise the action of specific invertebrate hormones. Therefore, the aim of the present study is to shift the focus from vertebrate type hormones and instead concentrate on the potential of exogenous invertebrate type hormones and their anthropogenic analogues to effect hormonally regulated processes in a model invertebrate, (the decapod crustacean, C.maenas). The specific endocrine mediated functions under investigation are moulting, vitellogenesis and locomotor activity.
The hormone being applied exogenously in this study is the ecdysteroid moulting hormone 20-hydroxyecdysone. The ecdysteroid moulting hormone ecdysone is produced endogenously in the Y organ (a paired epithelial endocrine gland) (Fingerman 1987) of decapod crustaceans. The Y organ converts dietary cholesterol into ecdysone and secretes it into the haemolymph. It is then transported to peripheral tissues where it is converted to the bioactive haemolymphatic form, 20-hydroxyecdysone (or b-ecdysone), titres of which increase just prior to moulting (Lachaise et al 1993). Ecdysteroids exert their effect by binding to an intracellular receptor protein within the target tissue. Synthesis of ecdysteroids by the Y organ is under inhibitory control from MIH (moult inhibiting hormone). MIH is a member of a different group of hormones, the neuropeptides, produced from the X-organ/sinus gland complex associated with the eyestalk in decapods. Ecdysteroid synthesis is also under stimulatory control from another steroid hormone, the sesquiterpenoid methyl farnesoate (MF), produced by the mandibular organ (Tamone and Chang 1993).
20-hydroxyecdysone, and the other ecdysteroids are not only involved in the moulting process but have also been shown to be involved in reproduction and vitellogenesis and may have a role in behaviour and rhythmic locomotory processes. It has been suggested that ecdysteroids may stimulate vitellogenesis (Fingerman 1997) and ovarian maturation and protein synthesis (Chan 1995, Oberdorster and Cheek 2001), although this is still poorly understood. Indeed, in some species, administration of ecdysteroids has inhibited vitellogenesis (Chang 1989). It appears that the ability of ecdysteroids to promote vitellogenesis in the hepatopancreas is species dependent (Loeb 1993) as is the need for ecdysteroids for the completion of vitellogenesis (Pinder et al 2000). Correlations between haemolymph ecdysteroid titres and vitellogenesis have been reported (Chang 1993, Okumura et al 1992, Young et al 1993a,b). However, it is still unclear as to whether ecdysteroids directly influence vitellogenesis, or that their levels during vitellogenesis are simply indicative of the corresponding stage of the moult cycle.
In order to identify any hormonally related disturbances in the processes of moulting, vitellogenesis and locomotor activity in crustaceans, it is important to understand the functioning of these systems under normal conditions.
Crustaceans must shed their exoskeleton (ecdysis) periodically for growth to occur, a process known as moulting. Ecdysis is in fact only a small part of the moult cycle, which can take up to a year or more and involves profound physiological and biochemical changes (Chang 1995).
Endocrine control of moulting.
During the moult cycle, circulating levels of ecdysteroids vary considerably. At postmoult, haemolymph titers of ecdysteroids are negligible and remain so throughout intermoult. Ecdysteroid levels increase dramatically in premoult and then drop steeply prior to ecdysis (Chang 1989). During intermoult, production and secretion of ecdysteroids by the Y-organ is under inhibitory control from MIH, produced by the x-organ/sinus gland complex, keeping circulating titers low. High affinity binding of MIH to Y organ membrane bound receptors has been demonstrated in C.maenas (Webster 1993). During early premoult, the Y-organ is freed from inhibition and ecdyteroid levels rise, triggering the latter stages of premoult, before dropping prior to ecdysis. Postmoult sees ecdysteroid production inhibited and haemolymph titers returned to basal levels. Removal of Y-organ inhibition by eyestalk ablation leads to a rapid and dramatic increase in the levels of circulating ecdysteroids and therefore to precocious moulting (Chang 1989,1995, Skinner 1985). This shortening of the moult cycle has been observed in many crustaceans (Chang 1989 1995, Skinner 1985).
The present study seeks to determine if such precocious moulting could be triggered by exogenous sources of ecdysteroids or compounds which mimic their action. This would be distinctly disadvantageous for field exposed crustaceans. Such individuals might prematurely enter into stages of the moult cycle whilst being subject to unfavourable environmental conditions or be at increased risk of predation. The present study will address this issue and C.maenas is to be exposed to exogenous sources of 20-hydroxyecdysone and the effect on moulting observed.
Female decapods develop a large number of heavily yolked eggs (oocytes) in the ovary. C.maenas for example, produces an average of 185,000 eggs in each reproductive cycle (Crothers 1967). The yolk, containing proteins, lipids and carbohydrates provides nourishment for the developing embryos and nauplii which must subsist on it for up to several weeks following hatching (Tom et al 1992). The process of yolk synthesis and deposition is termed vitellogenesis (Subramoniam 1999) and the major yolk protein that accumulates in the oocytes is vitellin (Vt) (Lee et al 1996). The primary translation product and precursor of vitellin is a high density lipoprotein called vitellogenin (Vg), present in the haemolymph of vitellogenic females. Vg is immunologically identical to Vt (Lee et al 1996). Quantitative and semiquantitative correlations between haemolymph Vg and ovarian Vt concentrations in developing oocytes and have been observed in many crustaceans (Adiyodi 1985, Okumura and Aida 2000). It is suggested that vitellogenin is synthesised in the ovary and also in extraovarian tissues (specifically the hepatopancreas) and transported in the haemolymph to the ovary, where it is converted to vitellin and incorporated as yolk globules into developing oocytes (Lee et al 1996, Chen and Chen 1994).
Endocrine control of vitellogenesis.
Vitellogenesis is under the strict control of a number of antagonisitic hormones. There are four main hormones involved in the control of vitellogenesis - the neuropeptide gonad inhibiting hormone (GIH), (also known as vitellogenesis inhibiting hormone -VIH), neuropeptide gonad stimulating hormone (GSH), the sesquiterpenoid methyl farnesoate (MF), and ecdysteroid(s). This study is concerned primarily with the role of 20-hydroxyecdysone in vitellogenesis and attempts to elucidate the effect of exogenous application of this hormone on Vg levels in female crabs.
There is evidence in the literature that in some crustaceans, ecdysteroids play a role in vitellogenesis. It has been suggested that high ecdysone titres related to premoult processes may promote the early stages of vitellogenesis (Skinner 1985). Ecdysteroids (ecdysone, 20-hydroxy ecdysone and ecdysteroid conjugates) have been detected in follicles, oocytes and embryos of several species of shrimp, crab and amphipod (Chang 1989), and the available evidence indicates that these ecdysteroids are sequestered into the ovary by binding to yolk precursor proteins (Subramoniam 2000). The specific function of these ovarian ecdysteroids is, however, unclear. Increased ecdysteroid titres in the haemolymph have been correlated with the progression of vitellogenesis in certain species, including C.maenas (Lachaise et al 1981), the spider crab Acanthonyx lunulatus (Chaix et al 1982) and the freshwater prawns Macrobrachium nipponense (Okumara et al 1992) and M.rosenbergii (Young et al 1993a). However, other studies have reported decreasing levels of haemolymph ecdysteroids during vitellogenesis eg: Panaeus monodon (Young et al 1993b). Young et al (1993a) suggest the differences observed may indicate that the roles of ecdysteroids in vitellogenesis in these two species differ. ie: ecdysteroids stimulate vitellogenesis in M.rosenbergii, but are not directly involved in P.monodon vitellogenesis.
Similar conflicting results are reported in studies where ecdysteroids are administered in vivo or in vitro, with the effect being either inhibitory or stimulatory (Chang 1993, Chang 1989). Clearly furthur research is required to determine the roles of ecdysteroids in vitellogenesis and the present study aims to determine the effect of exogenous ecdysteroids on vitellogenesis in the shore crab, be it inhibitory or stimulatory.
LOCOMOTOR ACTIVITY AND ENDOGENOUS RHYTHMICITY.
In addition to the processes of moulting and reproduction in C.maenas, the present study will investigate the hormonal control of locomotor activity in this species. At present, information on the hormonal regulation of locomotor activity in crustaceans is limited. Presented below is a general overview of C.maenas locomotor activity patterns relevant to the present research and a brief description of what is presently known about their hormonal regulation.
Locomotory activity patterns in C.maenas.
The locomotory activity exhibited by C. maenas is regulated by interlinked endogenous circadian and circatidal rhythms. The resultant locomotor pattern consists of peaks of activity around times of high tide (circatidal) overlaid with periods of increased activity at times of nocturnal high tides as compared to diurnal high tides (circadian) (Williams 1985). Essentially, the level of activity associated with a tidal peak is modulated by the circadian rhythm (Webb 1983). This rhythmicity in locomotor activity allows C. maenas to undertake daily migrations into inter-tidal areas where food and mating sites can be found. Increased activity at the time of nocturnal high tides also affords crabs increased foraging opportunities with limited risk of predation. Cessation of activity at low tide allows crabs to seek shelter, avoiding dessication and avian predation (Naylor 1985).
The characteristic pattern of locomotor activity is maintained in the laboratory under constant conditions for 4-6 days before starting to break down (Williams 1985) with crabs kept in normal light-dark, non-tidal conditions exhibiting a “daily rhythm” overlaid with a weak approximate tidal component (Webb 1983). Crabs kept for a month or more under constant conditions, whilst subject to constant illumination, showed a circadian rhythm with no tidal component (Webb 1983).
Under natural conditions, circadian/tidal rhythms are continually entrained (synchronised with predictable patterns of environmental change) by a number of exogenous variables (salinity, hydrostatic pressure, temperature, immersion and wave action) to ensure the expressed behaviour is in phase with the environmental regime (Bolt and Naylor 1986, Naylor 1985, Reid and Naylor, 1985). Under constant laboratory conditions, in the absence of environmental cues, the accuracy of endogenous free running rhythms slowly decreases and rhythmic patterns of behaviour become imprecise (Naylor 1985). Tidal rhythms in C. maenas can, however, be entrained in the laboratory by simulated tides with peaks of high salinity (Bolt and Naylor 1986), and subtle changes in temperature and pressure associated with tides in the normal habitat (Williams and Naylor 1969, in Naylor 1985). The entrained rhythmicity is then maintained when the animal is returned to constant conditions.
Endocrine control of locomotor activity and rhythmicity.
Little is presently known regarding the endocrine regulation of locomotor activity and endogenous rhythms in decapod crustaceans. Experiments involving eyestalk ablation identified the presence of an eyestalk factor responsible for the regulation of locomotor activity in crustaceans. Further research determined this factor to be a neuropeptide, termed neurodepressing hormone (NDH). NDH, secreted by the sinus gland, depresses the responsiveness of motor and sensory neurons and decreases the spontaneous firing of motor neurons (Fingerman 1987). It is thought to have a role in the modulation of circadian activity (Williams 1985, Arechiga et al 1974, 1979) and its release is rhythmic, reflecting the rhythmic activity patterns seen in crustaceans (Webb 1983). The cyclical nature of its release also suggests it is under the partial control of a biological clock mechanism elsewhere in the CNS (Naylor 1985). Removal of this inhibitory influence by eyestalk ablation results in prolonged heightened, arythmic locomotory activity in C. maenas (Williams 1985, Bolt and Naylor 1986). Eyestalk ablation has also been shown to abolish entrainability in C.maenas, as the ability to entrain to high salinity episodes in the laboratory is lost in eyestalkless crabs (Bolt and Naylor 1986). This is consistent with the view that the eyestalk neurosecretory complex is the site of a possible component of the crab’s physiological clock (Bolt and Naylor 1986).
The biogenic amine 5-HT has been shown to stimulate release of NDH and produces hyperglycaemia in crayfish, presumably by stimulating release of CHH from the sinus gland (Fingerman 1995). GABA (gamma amino butyric acid) has been shown to inhibit the release of NDH (Fingerman 1995). The regulation of release of these two peptide hormones has implications for locomotor activity in crustaceans. Locomotor activity may also be affected by the cardioexcitatory influence of 5-HT, dopamine and octopamine, released from the pericardial organ (Pinder et al 2000), which increase the frequency and amplitude of the heartbeat (Fingerman 1987).
Preliminary experiments conducted with C.maenas during the summer here in Plymouth have also suggested a possible role for ecdysteroid mediated perturbations to locomotor activity. Exposure to waterborne 20-hydroxyecdysone abolished the characteristic locomotor activity in several test animals under laboratory conditions. These preliminary experiments will be resumed with crabs being exposed to waterborne 20-hydroxyecdysone whilst contained within an actograph system designed to record locomotory activity over a period of 5-6 days.
In summary, the present study aims to determine the effect of waterborne exposure to the steroid moulting hormone 20-hydroxyecdysone on three endocrine mediated processes in C.maenas - moulting, locomotor activity and vitellogenesis.