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Co-ordination of responses involves the reception of stimuli and subsequent reaction. Exogenous signals include day length, lunar phases, tides, water levels in rivers as well as external salinity. Endogenous signals include levels of energy stores (adiposity) and plasma osmolality and pH. Responses are mediated by the neuroendocrine and endocrine signalling pathways which invoke the appropriate responses from specific cells and tissues.

In the case of salinity adaptation there are various local and circulatory signalling systems and agents which modulate the expression and function of ion and water transporters in eel tissues (Evans, 2002).

When teleosts are faced with an osmoregulatory challenge there are two hormonal response types; the first is fast and short-acting whilst the second is slow and long-acting. The first type offer an immediate response to the osmoregulatory stress but are often cleared from the system within

minutes (Takei and Hirose, 2002). They include oligopeptide hormones such as angiotensin II, arginine vasotocin, natriuretic peptides and urotensins which target specific epithelia where they modulate existing ion channels or

transporters by phosphorylation or dephosphorylation of key residues. The long-acting response hormones show gradual increases to elevated levels for prolonged periods of several hours or longer. This group incorporates

cortisol, growth hormone (GH), insulin-like growth factors (IGFs) and prolactin (PRL) and stimulate the synthesis of ion channels and transporters and induce structural reorganisation so that the animal can cope, long term, with its new environmental salinity.

adenylate cyclase activating polypeptide, corticotropin-releasing hormone, somatolactin release-inhibiting hormones and thyrotropin releasing hormone. The tiered organisation of these systems is shown in context with their secretion sites, pathway induction and site of action (Figure 2.6.a).

Figure 2.6.a. Hierarchical overview of the response to sensory input via the hypothalamo-hypophysial axis resulting in an endocrine cascade to peripheral glands and tissues. CRH: corticotropin-releasing hormone, U-I: urotensin-I, AVT: arginine vasotocin, TRH: thyrotropin-releasing hormone, GnRH: gonadotropin-releasing hormone, GHRH: growth hormone-releasing hormone, GHIH: growth hormone-inhibiting hormone, PRH: prolactin-releasing hormone, PIH: prolactin-inhibiting hormone, ACTH: adrenocorticotropic hormone, TSH: thyroid stimulating hormone, GTH-I and -II: gonadotropin-I and –II, GH: growth hormone, PRL: prolactin, SL: somatolactin, MSH: melanophore stimulating hormone, MCH: melanin concentrating hormone. Adapted from Takei and Loretz, 2006.

One of the principal sites of hormone secretion is the pituitary gland, which is subdivided into two functionally and anatomically distinct sections: the anterior pituitary and the posterior pituitary (neurohypophysis) (Figure 2.6.b.). The anterior pituitary (adenohypophysis) originates from the

nasopharangeal epithelium and is subdivided into the rostral pars distalis and proximal pars distalis. It is responsible for the secretion of gonadotropin-I and –II (orthologous to follicle stimulating hormone and luteinising hormone

respectively), thyroid stimulation hormone, growth hormone, prolactin, adrenocorticotropic hormone and somatolactin. The posterior pituitary is derived from neural tissue and comprises the pars intermedia and pars nervosa and secretes the neuroendocrine hormones; melanin concentrating hormone, arginine vasotocin and isotocin.

responsible for urotensin-I and –II secretion, reviewed in Kobayashi et al (1986). Additionally, atrial natriuretic peptide, a seawater adapting hormone (Takei and Balment, 1993), is secreted by the heart whose release is

stimulated by increases in plasma osmolality in teleosts (Kaiya and Takei, 1996) and by cardiac stretch in mammals (Farrell, 1999). Upon exposure to seawater the blood volume in the eel decreases (Kaiya, 1996b), which would normally inhibit ANP secretion in mammals. In teleosts, however, the

increase in plasma osmolality has an overriding effect and ANP secretion increases. This in turn causes a dose dependent decrease in angiotensin II levels which inhibits both drinking (Tsuchida, 1998) and Na+/Cl- absorption by the intestine (Loretz, 1997). This appears, at first, to be counter intuitive as the eel in seawater must drink copiously to ensure adequate water uptake. Indeed, upon initial transfer to seawater the eel elevates its drinking rate substantially, a response which is initiated by external chloride receptors as it occurs prior to increases in plasma osmolality (Takei et al., 1998). Drinking rapidly abates, however, as the influence the plasma osmolality-stimulated increase in ANP secretion overrides these mechanisms. Increased ANP secretion and the associated anti-dipsogenic effects are transient, however, decreasing after 1-2 hours post seawater transfer (Kaiya and Takei, 1996b), at which point drinking rates become elevated once again. Therefore this system allows the eel a period of respite, during which, alimentary ion transport systems can adapt to seawater.

Following the work by Utida et al the traditional view was that, amongst teleosts, cortisol was the seawater adapting hormone and prolactin was the

freshwater hormone (Utida et al., 1972). In the freshwater adapted killifish for example, Fundulus heteroclitus, plasma cortisol levels peak 1 hour post

transfer to seawater, which coincides with the highest plasma Na+ _level (Marshall, 1999). More recent studies have now shown that cortisol may actually have a dual role as it is also implicated in ion uptake, whilst seawater acclimation also involves the growth hormone (GH)/ insulin like growth factor I (IGF-I) axis (McCormick, 2001). Salmonids treated long term with GH

(Komourdjian et al., 1976) or transgenically over expressing the hormone (Saunders et al., 1998) show elevated levels of salinity tolerance. Although

larger fish will have an inherently greater salinity tolerance, the same effect has been noted just 48 hours following growth hormone injection, before any size effects could be implicated (Bolton et al., 1987). Several salinity related physiological changes induced by GH and IGF-I have been noted. Prunet et al (1994) found GH treated Atlantic salmon exhibited increased numbers of secretory mitochondria rich and accessory cells in the gill. In vivo Na+,K+-

ATPase expression and activity are also raised in the gill mitochondria rich cells (Mancera and McCormick, 1998; Sakamoto et al., 1997), as is the Na+- K+-2Cl- cotransporter (Pelis and McCormick, 2001). Examining the system from the other side, elevated levels of GH and IGF-I have been shown in salmonids following salinity challenge (Sakamoto et al., 1993). Pertinent to the current study, however, is the lack of evidence which relates GH and IGF-I to osmoregulation within the anguillid eels. In cultured pituitary cells, an osmotic challenge had no effect on GH secretion levels (Suzuki et al., 1991; Suzuki et al., 1990). Additionally, eels have also been shown to survive in seawater without a pituitary, and therefore without growth hormone (Takei and Hirose, 2002).

In addition endocrine and neuroendocrine systems discussed, there must be also be pathway for sensing and coordinating environmental cues with endogenous factors to bring about eel silvering at a time to maximise reproductive success. Furthermore the eel must be able to react to additional environmental signals which dictate the timing of migration in a way that synchronises their arrival at the spawning grounds. One such mechanism for co-ordinating these responses in the eel could involve leptin (Zhang et al., 1994), which has been characterised as a signal peptide relating somatic energetic status to the reproductive system. Early leptin research focussed on the function of this adipocyte secreted peptide in relation to appetite

regulation (Campfield et al., 1995). However, knockout mice, either incapable of producing leptin (ob/ob)or lacking the leptin receptor (db/db) were found to

signal peptide (Chehab et al., 1996). The first reported mechanism of interaction with reproductive pathways in mice was shown by direct injection of leptin which induced an increase in levels of circulating LH (Barash et al., 1996). Likewise, leptin has since been shown to influence follicle stimulating hormone in a similar way (Yu et al., 1997).

Leptin has recently been cloned in a number of teleost fish (Huising et al., 2006; Kurokawa et al., 2005) but the process of understanding its role is in its infancy. Some of the functions of teleost leptin appear to be conserved with other chordates, but the mechanisms involved remain unclear. In mammalian systems leptin appears to be antagonistic to neuropeptide Y, but this relationship is not immediately discernable in teleosts. Ammar et al

(2000) showed that leptin is an appetite suppressant and stimulant of reproductive behaviour whilst neuropeptide Y stimulates eating and suppresses reproductive behaviour. The opposing roles of these two peptides is further highlighted by the direct inhibitory action of leptin upon neuropeptide Y synthesis in the hypothalamic arcuate nuclei (Baskin et al., 1999). As mentioned previously, leptin has been shown to cause an increase in LH secretion in mice but in teleosts, neuropeptide Y has also been shown to stimulate LH release. Two parallel studies, one on goldfish (Kah et al., 1989) and the other on rainbow trout (Bernard et al., 1989) both showed that porcine neuropeptide Y treatment of in vitro pituitary cell cultures induced a

dose dependent increase in LH release. These findings are in agreement with a more recent in vivo study of seabass (Dicentrarchus labrax), which also

showed that neuropeptide Y causes an increase in LH secretion (Cerda- Reverter et al., 1999). The effects of neuropeptide Y in this case, however, were dependent on nutritional state; stimulation of LH secretion was only seen in chronically fasted animals whilst the effects were suppressed in fed

animals. One of the first leptin studies in teleosts treated cultured pituitary cells from carp (Cyprinus carpio) with purified mouse leptin and demonstrated

an increase in secretion of LH; the amplitude of the response was dependent on the sexual maturation stage (Peyon et al., 2001). Therefore, at first glance the actions of leptin upon LH secretion appear to be complementary to those of neuropeptide Y. The situation is still unclear, however, as the stimulatory

effect on LH secretion seems to be nutritionally dependent for neuropeptide Y and developmentally dependent for leptin. These pathways are candidates for the regulation of sexual development in relation to energy stores in the eel, and may provide answers as to why, as in mammals, the onset of puberty is size not age dependent (Foster and Nagatani, 1999).