Le Loup ou Bar Dicentrarchus labrax est une espèce euryhaline dont les femelles pondent généralement en eau de mer. Pendant son cycle de vie, des migrations vers les estuaires et les lagunes, peuvent exposer très tôt les jeunes stades à des variations de salinité. Les mécanismes de l'osmorégulation sont bien connus chez les prélarves, les larves et les adultes de D. labrax en eau de mer (EM, 38) et en eau de mer diluée (EMD, 5), cependant les possibilités d'osmorégulation et leurs mécanismes sont inconnus chez les embryons. Le but de cette étude a été d'évaluer la mise en place de la fonction osmorégulatrice chez les embryons de D. labrax.Tout d'abord le développement embryonnaire des différents sites osmorégulateurs a été décrit, en se focalisant sur le tube digestif, en incluant le pharynx et les premières fentes branchiales. La formation de ces structures débute au stade 12 somites (S) et a été décrite jusqu'à l'ouverture de la bouche, 5 jours après l'éclosion.En second lieu, le lieu et la cinétique d'apparition des premières cellules osmorégulatrices, les « ionocytes » ont été recherchés. Ces cellules ont été identifiées au stade 12S sur la membrane de la vésicule vitelline et au niveau des premières fentes branchiales et du tube digestif primitif au stade 14S. La fonctionnalité de ces cellules a été étudiée grâce à des immunomarquages des principales protéines transmembranaires impliqués dans l'osmorégulation [l'ATPase Na+/K+ (NKA), le cotransporteur Na+/K+/2Cl- (NKCC) et le canal à chlore (CFTR)], et avec une étude ultrastructurale. Des ionocytes potentiellement fonctionnels sont présents à partir du stade 25S au niveau de la membrane de la vésicule vitelline et du tube digestif primitif, mais les ionocytes des premières fentes branchiales ne sont pas totalement fonctionnels à l'éclosion. L'existence d'un phénomène de boisson passive qui permettrait la régulation hydrique chez D. labrax est envisagé.Finalement, l'osmorégulation embryonnaire existant en EM et en EMD a été étudiée. Des mesures nanoosmométriques des fluides embryonnaires indiquent une capacité à hyper- et hypo-osmoréguler. Cependant, en EMD, des analyses en qRT-PCR et des immunomarquages de NKA, NKCC et CFTR révèlent que les mécanismes de l'hyper-osmorégulation peuvent limiter les pertes ioniques mais ne sont pas suffisamment efficaces pour permettre une acclimatation totale à l'EMD à ce stade très précoce. / The European sea bass Dicentrarchus labrax is a euryhaline species which usually spawns in seawater. Due to its life cycle that includes migrations to lagoon and estuaries, young stages can be exposed early to salinity variations. Osmoregulatory patterns are well known in prelarvae, larvae and adults D. labrax in seawater (SW, 38) and in dilute seawater (DSW, 5), but the possibility and mechanisms of embryonic osmoregulation are still unknown. The goal of this study was to investigate the occurence of the omoregulatory function in the embryos of D. Labrax.First, the embryonic development of the different osmoregulatory sites was described, focusing on the digestive system including the pharynx and the first gill slits. The formation of these structures is initialized at stage 12 somites (S) and was described throughout the opening of the mouth five days after hatching.Secondarily, the time and the location of the occurrence of the first osmoregulatory cells, the ionocytes were followed. These cells were identified at stage 12S on the yolk sac membrane and at stage 14S in the first gill slits and in the posterior primitive gut. The functionality of these cells was studied, using immunostaining of the main ionic transporters involved in osmoregulation [the Na+/K+ ATPase (NKA), the Na+/K+/2Cl- cotransporter (NKCC) and the chloride channel (CFTR)], and through ultrastructural investigations. Potentially functional ionocytes are present from stage 25S in the yolk sac membrane and in the gut, but gill slits ionocytes are not fully functional at hatching. Passive drinking is suspected to regulate water balance in D. labrax.Finally, the embryonic osmoregulation in SW and DSW was investigated. Nanoosmometry measurements of the embryonic fluids demonstrated some capabilities of hyper- and hypo-osmoregulation. However, in DSW, qRT-PCR and imunostaining of NKA, NKCC and CFTR, reveal that hyper-osmoregulatory mechanisms can only limit ion loss but are not efficient enough to allow a full acclimation at this early life stage.

Le loup Dicentrarchus labrax est un téléostéen euryhalin qui effectue des migrations saisonnières de la mer vers les lagunes et les estuaires où la salinité est très variable et peut changer rapidement. Les mécanismes d'osmorégulation sont bien connus chez les téléostéens, alors que le rôle de l'osmodétection dans leur mise en place reste actuellement largement méconnu. Le but de cette étude a été d'améliorer les connaissances sur l'osmodétection et sur la mise en place des mécanismes d'hyper-osmorégulation (à court et long terme).Tout d'abord, le canal calcique “Transient Receptor Potential Vanilloid 4” ou TRPV4, candidat osmodétecteur, a été étudié chez des loups adaptés à l'eau de mer (EM) ou exposés à l'eau douce (ED) durant des temps différents. Au niveau branchial, le TRPV4 ne semble pas impliqué dans l'osmodétection au niveau des ionocytes, mais plutôt dans l'absorption de Ca2+ par les chondrocytes, notamment en ED. Il pourrait avoir un rôle dans l'osmodétection systémique au niveau de l'hypophyse et dans l'absorption de Ca2+ dans le rein. Sa forte expression en EM dans les cellules rénales réabsorbant de l'eau suggère que le TRPV4 est impliqué dans la régulation de leur volume cellulaire.En second lieu, le canal à chlore ClC-3, candidat osmodétecteur et potentiellement impliqué dans les mécanismes de diminution régulatrice du volume cellulaire (RVD), a été étudié dans les mêmes conditions expérimentales que précédemment. Ce canal a été localisé dans la membrane basolatérale des ionocytes des branchies (EM et ED) et des tubes collecteurs du rein (en ED ; pas immunodétecté en EM). Il pourrait jouer un rôle dans la protection des cellules contre un choc hypotonique, et donc dans la RVD en EM dans ces deux organes. En ED, la localisation du ClC-3 suggère fortement sa participation dans l'hyper-osmorégulation au niveau des branchies et du rein.Enfin, un protocole d'isolement des ionocytes branchiaux a été mis au point ; il permettra de poursuivre et d'approfondir les travaux sur l'osmodétection cellulaire. L'évolution au cours du temps du volume cellulaire d'un ionocyte a pu être évaluée après un choc hypotonique. De futures expérimentations permettront d'évaluer le lien entre l'osmodétection, les osmodétecteurs et les effecteurs osmotiques. / The European sea bass Dicentrarchus labrax is a euryhaline teleost that migrates seasonally from the sea to lagoons and estuaries, where salinity is very variable and changes rapidly. Osmoregulatory mechanisms are well known in teleosts, while the role of osmosensing in their set-up is yet poorly understood. The objective of this study was to improve the understanding of osmosensing and of its relations with hyper-osmoregulatory mechanisms in sea bass over short- and long-term salinity exposures.First, the Transient Receptor Potential Vanilloid 4 (TRPV4) Ca2+-channel, a potential osmosensor, has been studied in fish adapted to sea water (SW) or exposed to fresh water (FW) for various times. The TRPV4 did not seem to be involved in osmosensing in mitochondrion-rich cells (MRC) in gills. It may be involved in systemic osmosensing and in Ca2+ uptake by branchial chondrocytes and by renal collecting ducts in FW. This channel may also have a role in cell volume regulation of kidney cells allowing water reabsorption.Secondly, the ClC-3 chloride channel, another potential osmosensor and potentially involved in regulatory volume decrease (RVD) mechanisms, has been studied in fish under similar conditions. This channel has been localized in the basolateral membrane of ionocytes in gills (SW and FW) and kidney (FW – in SW, expressed but not immunodetected). It might be involved in cell protection against hypotonic shock and thus in RVD in both organs in SW. In FW, its localization strongly suggests a role in hyper-osmoregulation in gills and kidney.Finally, a protocol to isolate MRCs has been developed; it has been used to study cell osmosensing and it will be used on future studies. The changes in cell volume following a hypotonic shock have been evaluated. Future experiments will allow a better understanding of the relations between cell osmosensing, osmosensors and effectors.

Les mangroves sont de plus en plus mentionnées comme outil de bioremédiation potentiel dans le traitement des eaux usées (EU). Actuellement, les effets des rejets d’EU sur la macrofaune, et plus particulièrement sur les crabes de mangrove, ne sont pas clairs. Ces espèces sont dites ingénieurs de cet écosystème, notamment grâce à leur activité de bioturbation qui permet, entre autres, l’infiltration des EU dans le sédiment via leurs terriers. L’objectif ont donc été d’étudier l’impact du rejet d’EU domestiques sur la physiologie (osmorégulation, métabolisme et balance oxydative) de 3 espèces de crabe (2 Sesarmidae et 1 Ocypodidae) par une approche combinant expérimentations en laboratoire et sur le terrain en utilisant un site pilote expérimental sur l’île de Mayotte. Ces crabes qui vivent dans la zone intertidale ont un mode de vie bimodal et font fréquemment face à des salinités variables. Ils sont de bons hyper-hypo-osmorégulateurs et sont adaptés à cette vie à l’interface entre terre et eau aussi bien au niveau de la régulation ionique que de la respiration. Les résultats indiquent que la densité des terriers diminue dans les zones d’écoulement des EU et que la communauté des espèces est modifiée avec la dominance de Parasesarma guttatum (PG) qui n’est pas une espèce bioturbatrice. Les EU induisent donc une modification potentielle du fonctionnement de l’écosystème. PG diminue son métabolisme alors que les deux autres espèces étudiées l’augmentent significativement. Immergées dans les EU, les trois espèces étudiées présentent des atteintes de la fonction osmorégulatrice (activité de la Na+/K+-ATPase et épaisseur d’épithélium branchiale) et de la balance oxydative (formation d’espèces réactives de l’oxygène dans l’hémolymphe et enzymes antioxydantes des branchies) en laboratoire mais des effets moins marqués sont observés chez les crabes maintenus in situ dans des terriers artificiels. Les biomarqueurs étudiés peuvent ainsi être utilisés pour mesurer l’état physiologique des crabes soumis à des rejets d’EU domestiques. Ces atteintes qui entraînent des coûts métaboliques supplémentaires peuvent mener à la réduction de leur fitness, contribuant à expliquer les observations écologiques. De plus, les résultats montrent que les crabes violonistes sont les plus sensibles, suivis des deux Sesarmidae alors que PG semble mieux adapté pour éviter les EU. Si aucun dysfonctionnement majeur n’a été observé à l’échelle de l’écosystème jusqu’à présent, il convient de maintenir un suivi régulier de ces espèces, en tenant compte de leur spécificité en termes d’activité bioturbatrice et de santé physiologique. / Mangroves are increasingly proposed as a bioremediation tool for wastewater (WW) treatment. However, this practice can impact mangrove crabs which are key engineer species of the ecosystem through their bioturbation activities. Their burrows are directly involved in the bioremediation process allowing WW infiltration in the sediment. This study aimed to determine the effects of WW on the physiology (osmoregulation, bioenergetics, oxidative balance) of 3 species of crabs (2 Sesarmidae and 1 Ocypodidae) with laboratory and in situ experiments (burrow density and caging experiment in an experimental area with controlled WW releases on a mangrove located on the island of Mayotte). These crabs inhabit the intertidal area of variable salinity with a bimodal life (aquatic and terrestrial). They are good hyper-hypo-osmoregulators and well adapted to terrestrial life both in terms of osmotic and aerial breathing capacities. Burrow density decreases in flat areas where WW flows and crab community is altered with a marked dominance of Parasesarma guttatum (PG) (a species with no bioturbation activity). This change may induce drastic alterations of the ecosystem functioning. The bioenergetic response of PG is totally different from the other studied species. PG decreases its metabolic rate in WW but the other species have increased metabolic activity. Moreover, after laboratory exposure the 3 species show impairments in their osmoregulatory capacity (Na+/K+-ATPase activity and epithelium gill thickness) and oxidative balance (reactive oxygen species formation in haemolymph and antioxidant enzyme activity in gills) due to WW exposure in laboratory conditions. In situ, encaged crabs showed a similar but reduced pattern. These effects could decrease their fitness and may also explain the observed ecological changes. The biomarkers used in this study may be a useful tool to monitor crab populations. Moreover, our results show that fiddler crabs are the most sensitive to WW followed by other Sesarmidae. PG seems better adapted to avoid WW exposure. Even if no major dysfunction is observed at the ecosystem level yet, WW release should be carefully monitored nevertheless with an emphasis on crab bioturbation activity and their physiological health according to species sensitivity.

All these findings can help us to elucidate the mechanisms for the fish to detect changing osmotic conditions and transform signals to osmoregulatory responses. / In the first part of the study, PRL and PRL-releasing peptide (PrRP) cDNAs have been isolated from euryhaline silver sea bream. The PRL cDNA consists of 1360 bp encoding 212 amino acids whereas the PrRP cDNA contains 631 bp encoding prepro-PrRP with 122 amino acids. PRL mRNA was uniquely expressed in sea bream pituitary but PrRP mRNA was expressed in a variety of tissues. Expression levels of both PRL and PrRP mRNA have been examined in sea bream adapted to different salinities (0, 6, 12, 33 and 50 ppt). In pituitary, both PRL and PrRP mRNA were synchronized in their expression, being significantly higher in fish adapted to low salinities (0 and 6 ppt), but the expression profile of hypothalamic PrRP in different salinities was different. These data suggested that PrRP may possibly act as a local modulator in pituitary rather than a hypothalamic factor for regulating pituitary PRL expression in silver sea bream. / In the second part of the study, silver sea bream abruptly transferred from 33 to 6 ppt exhibited a remarkable pituitary PRL secretion following closely with the temporal changes in serum osmolality and ion levels. In order to investigate the direct effect of extracellular osmolality to pituitary PRL secretion, sea bream pituitary cells were dispersed and exposed to a medium with reduced ion levels and osmolality. PRL released from pituitary cells was found to be significantly elevated. In hyposmotic exposed anterior pituitary cells, cell volume exhibited a 20% increase when exposed to a medium with a 20% decrease in osmolality. These enlarged pituitary cells did not shrink until the surrounding hyposmotic medium was replaced, a phenomenon suggesting an osmosensing ability of silver sea bream PRL cells for PRL secretion in response to a change in extracellular osmotic pressure. / In the third part, olfactory rosette in the nasal cavity was surgically removed from silver sea bream adapted to 6 ppt and 33 ppt and mRNA expression of PRL and PrRP in silver sea bream were measured. The elevated pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish were abolished by this olfactory lamellectomy. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change but those in 33 ppt-adapted fish increase significantly after olfactory lamellectomy. These data suggest a possible osmosensing role of the olfactory system for regulation of PRL expression during hypo-osmotic acclimation of the fish. Besides, calcium-sensing receptor (CaSR) was cloned and its mRNA expression in olfactory system, as shown in other fish species previously, was investigated. However, no CaSR expression could be detected in olfactory rosette and nerve but its expression was demonstrated in osmoregulatory tissues and brain. There was no significant difference in CaSR mRNA expression in pituitary, kidney and anterior intestine of fish adapted to different salinities. These studies could not provide conclusive evidence to correlate CaSR with osmosensing in silver sea bream. / The present study used silver sea bream (Sparus sarba ) as a euryhaline fish model to investigate the regulation of prolactin (PRL) expression and secretion in fish adapted to different salinities. / Kwong, Ka Yee. / Adviser: Norman Y. S. Woo. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3248. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 154-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

A (Na+,K+)-ATPase presente no tecido branquial dos crustáceos osmorreguladores é um componente essencial de seu sistema de regulação iônica e osmótica. Esta enzima também apresenta um papel relevante no processo de excreção ativa de NH4+ através do tecido branquial dos crustáceos. Uma fração microsomal rica em (Na+, K+)ATPase foi preparada por centrifugação diferencial a partir de um homogeneizado do tecido branquial de Callinectes ornatus. A centrifugação em gradiente de sacarose revelou a presença de um unico pico de proteina com atividade ATPase, mas a eletroforese em gel de poliacrilamida em condições desnaturantes revelou a presença de várias bandas protéicas. O uso do anticorpo monoclonal 5 contra a subunidade da (Na+, K+) ATPase, revelou a presença de uma única banda proteica de 110 kDa com atividade (Na+, K+)?ATPase. A (Na+, K+) ATPase hidrolisou o PNPP (V= 52,0 ± 2,0 U/mg e K0,5 = 1,1 ± 0,1 mM) através de interações sítio-sítio (nH= 1,6). A modulação da enzima pelos íons magnésio (V= 52,3 ± 1,3 U/mg e K0,5 = 1,1 ± 0,05 mM), potássio (V= 51,4 ± 1,5 U/mg e K0,5 = 2,3 ± 0,1 mM) e amônio (V= 56,7 ± 2,6 U/mg e K0,5 = 9,8 ± 0,4 mM) ocorreu através de interações sítio-sítio. Os íons sódio atuaram como inibidores da atividade K+-fosfatase da enzima (Ki= 1,7 ± 0,1 mM) e a ouabaína inibiu cerca de 80% a atividade PNPPase independentemente da presença de íons amônio. A (Na+, K+) ATPase hidrolisou o ATP de acordo com cinética de Michelis-Menten, com KM= 0,16 0,01 mM e V= 116,3 5,6 U/mg, enquanto a modulação da atividade da enzima pelos íons magnésio (V= 111,0 ± 5,4 U/mg e K0,5= 0,54 ± 0,03 mM), sódio (V= 110,6 ± 5,3 U/mg e K0,5= 6,3 ± 0,3 mM), potássio (V= 116,0 ± 5,5 U/mg e K0,5= 1,5 ± 0,1 mM) e amônio (V= 173,3 ± 5,4 U/mg e K0,5= 5,4 ± 0,3 mM) ocorreram através de interações sítio-sítio. Também foi observado que na presença de concentrações crescentes de ions amonio, a estimulação da atividade (Na+,K+)-ATPase pelo íons potássio acarretou um aumento de 50% na atividade específica da enzima. A ouabaína inibiu cerca de 86% a atividade (Na+,K+)-ATPase com Ki= 74,5 ?M, sugerindo a presença de 14% de fosfatases e/ou outras ATPase contaminantes. Este é o primeiro trabalho onde se observa uma estimulação sinergística da atividade K-fosfatase da (Na+,K+)-ATPase de crustáceo pelos íons potássio e amônio. Os resultados cinéticos obtidos para a (Na+,K+)-ATPase branquial de Callinectes ornatus, analisados em conjunto com os já descritos para outras espécies de crustáceos poderão abrir novas perspectivas em relação ao papel dessa enzima na adaptação fisiológica-bioquímica, bem como para a sobrevivência desses animais em diferentes ambientes. / (Na+, K+)-ATPase present on branchial tissue osmoregulatory crustaceans is an essential component of their osmotic and ionic regulation system. Apparently, this enzyme also have a relevant role in the active excretion de NH4+ through the branchial crustacean tissue. A (Na+, K+) ATPase-rich microsomal fraction was prepared by differential centrifugation from Callinectes ornatus homogenized branchial tissue. The sucrose gradient sucrose centrifugation showed the presence of a single protein peak with ATPase activity, and SDS-PAGE revealed the presence of several proteins bands. The use of the 5 monoclonal antibody, against the ? subunit, revealed the presence of a unique protein band of 110 kDa corresponding to the (Na+, K+) ATPase. (Na+, K+) ATPase hydrolyzed the PNPP (V= 52.0 2.0 U/mg and K0.5= 1.1 0.1 mM) through the site-site interactions (nH= 1.6). The modulation of (Na+, K+) ATPase by magnesium (V= 52.3 1.3 U/mg and K0.5= 1.1 ? 0.05 mM), potassium (V= 51.4 1.5 U/mg and K0.5= 2.3 0.1 mM) and ammonia ions (V= 56.7 2.7 U/mg and K0.5= 9.8 ? 0.4 mM) followed cooperative kinetics. However, sodium ions inhibited PNPPase activity of (Na+, K+)?ATPase with Ki= 1.7 0.1 mM. Ouabain also inhibited up to 80% the total activity PNPPase independent of the presence of ammonium ions. The hydrolysis of ATP by (Na+, K+) ATPase followed Michaelis-Menten kinetics with Km= 0.16 0.01 mM and V= 116.3 5.6 U/mg, while enzyme modulation by magnesium (V= 111.0 5.4 U/mg and K0.5= 0.54 0.03 mM), sodium (V= 110.6 5.3 U/mg and K0.5= 6.3 0.3 mM), potassium (V= 116.0 5.5 U/mg and K0.5= 1.5 ? 0.1 mM) and ammonium ions (V= 173.3 . Interestingly, the stimulation of (Na+, K+)-ATPase activity by potassium ions in the presence of increasing concentration of ammonium ions to K+ resulted in a 50% higher specific activity. Ouabain inhibited approximately 86% the activity (Na+, K+) ATPase with (Ki= 74.5 M), suggesting the presence of about 14% of phosphatases and/or other ATPases. This is the first work showing synergistic stimulation of crustacean (Na+, K+) ATPase by potassium and ammonium ions when PNPP is used a substrate. The results reported herein for Callinectes ornatus branchial (Na+, K+) ATPase might open new perspectives concerning the physiological adaption and the survival of these animals in different environmental.

Inanga (Galaxias maculatus) is an amphidromous fish species that is able to successfully inhabit a variety of salinities. Using an integrated approach this thesis has characterised for the first time the physiological characteristics that facilitate acclimation in inanga. Structural studies using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM) revealed freshwater-acclimated inanga have a high density of apical pits and freshwater-type mitochondria-rich cells (MRCs) that can facilitate ion absorption from the hypo-osmotic environment. In seawater, inanga remodel their gills by increased proliferation of seawater-type MRCs to facilitate ion secretion in the hyper-osmotic environment. Concentration-dependent sodium (Na+) kinetic analysis revealed that at a whole body level, inanga regulate Na+ using a saturable, high affinity, low capacity uptake system which makes them extremely adept at extracting Na+ from very dilute freshwater environments. In fact inanga displayed an uptake affinity (Km) of 52 ± 29 µM, which is one of the lowest ever recorded in freshwater fish. The sodium/potassium ATPase transporter (NKA) is central to Na+ regulation within the gill. In high salinties inanga displayed increased NKA activity (6.42 ± 0.51 µmol ADP mg protein-1 h-1) in an effort to excrete the excess Na+, diffusively gained from the hyper-osmotic environment. This increase in NKA was most likely a reflection of the proliferation of NKA-containing MRCs. The NKA activities seen in freshwater- and 50% seawater-acclimated inanga were similar (2.54 ± 0.19 and 2.07 ± 0.22 µmol ADP mg protein-1 h-1 respectively) to each other suggesting the inanga gill is capable of supporting ion regulation in brackish waters without a significant increase in NKA activities, and the energetically-expensive changes in gill structure and function that accompany such a change. Molecular investigation of NKA isoform expression using quantitative PCR (qPCR) showed that inanga displayed salinity-induced changes in the expression of the three α NKA isoform variants investigated. Isoform α1a exhibited a pattern consistent with an important role in freshwater, confirming results from other fish species. While it is generally accepted that α1b isoform is the predominant NKA isoform in seawater, inanga did not display this pattern with a freshwater dominance seen. None of the salinity-induced changes could quantitatively explain the increased NKA activity in seawater suggesting that different isoforms may convey different activities, that there is also regulation of NKA at a post-transcriptional level, and/or other isoforms or subunits may have a significant role. The importance of the osmoregulatory hormone cortisol and prolactin is widely accepted and inanga were treated with cortisol, prolactin and a combination of the two in an effort to further elucidate their role. NKA activity and NKA isoform expression were assessed but no specific patterns were deduced, except for a decrease in both NKA activity and isoform expression in 100% seawater-acclimated inanga treated with cortisol and prolactin. The reasons for this decrease were not evident, although the impact of stress induced by the injection protocol was likely to be a confounding factor. The development of a new confocal-based technique in this study was able to describe, for the first time, intracellular sodium levels ([Na+]i) as a function of salinity in an intact euryhaline fish gill cell. Using the fluorescent Na+ indicator dye CoroNa Green this study demonstrated the ability of inanga gill cells to maintain [Na+]i in the face of environmental change. Freshwater-acclimated inanga displayed basal [Na+]i of 5.2 ± 1.8 mM, with 12 ± 2.3 mM and 16.2 ± 3.0 mM recorded in 50% seawater- and 100% seawater-acclimated cells, respectively. Low [Na+]i is advantageous in hypo-osmotic environments as it provides a gradient between the cell and the blood which is essential for generating electrochemical gradients cell volume regulation and other cellular homeostatic mechanisms. A slightly elevated [Na+]i seen at the higher sanities would help minimise the diffusive gradient for passive influx from the environment which would be of benefit in hyper-osmotic environments. Upon salinity challenge 50% seawater cells were equally adept at maintaining a constant [Na+]i at any salinity, suggesting these cells are have the necessary constituents to regulate Na+ in both lower and higher salinities. This novel LSCM approach is advantageous relative to existing transport models as it will allow the observation of cellular ion transport in real time, within a native filament structure displaying functional interaction of different cell types. The extreme ion uptake characteristics of the inanga and their amenability to in situ confocal-based studies demonstrated in this study, confirm inanga as a valuable model species for future research.

Salinity and oxygen availability have long been recognised as important factors influencing animal physiology and therefore species distribution. The maintenance of appropriate cellular ion levels is critical for many essential physiological processes, but at the same time is energetically expensive. Since hypoxia is likely to impose aerobic limitations for ATP generation, the maintenance of salt and water homeostasis could be at risk during hypoxia. The amphidromous inanga (Galaxias maculatus) is well known for its salinity tolerance and its life cycle that involves several salinity related migrations. During these migrations inanga also frequently encounters hypoxic waters, and therefore must maintain energy homeostasis when aerobic metabolism may be compromised. The present study has investigated behavioural, physiological, biochemical and molecular mechanisms by which inanga tolerate changes in salinity and hypoxia. After 14 days of acclimation to salinities ranging from freshwater to 43‰, inanga showed physiological acclimation. This was evident by no changes in metabolic rates or energy expenditures through this salinity range. Energy balance seemed to be tightly and efficiently controlled by changes in the proportion of protein and lipids used as energy substrate. No mortalities and only minor changes in plasma osmolality also indicated salinity acclimation. The remarkable osmoregulatory capacity of inanga was also evidenced after a seawater challenge. The osmotic balance of inanga was only disrupted during the first 24 hours after the challenge, evidenced by an increase in plasma osmolality and plasma Na+, and a decrease in muscle water content. These physiological changes were correlated with changes at the molecular level. Different isoforms of the catalytic subunit of the Na+,K+-ATPase (NKA) were isolated, partially sequenced and identified in inanga. Phylogenetic analysis grouped inanga isoforms (α-1a, α-1b, α-1c) with their respective homologues from salmonids. Patterns of mRNA expression were also similar to salmonids, with α-1a being downregulated and α-1b being up-regulated following seawater challenge. Previous to this study, NKA isoform switching was reported to occur only in salmonids and cichlids. The presence of NKA subunits that change with environmnetal salinity in inanga indicates that this isoform switching phenomenon is much more widespread among teleost lineages than previously thought. Aiming to elucidate the hypoxia tolerance of inanga, oxygen consumption rate as a function of decreasing external PO2 was evaluated. At no point did inanga regulate oxygen consumption, suggesting that this species is an oxyconformer. This is the first robust demonstration of the existence of oxyconforming in fish. Evaluation of the scaling relationship between oxygen consumption and fish size in normoxia, showed that the exponent of this relationship fell within the range previously reported for fish. However, in hypoxic conditions the scaling relationship was less clear suggesting different size-related mechanisms for tolerating hypoxia. Analysis of the aerobic and anaerobic metabolism of small and large fish, showed that smaller inanga were able to sustain aerobic metabolism for longer than larger inanga, which instead relied on anaerobic metabolism for extending their survival. This knowledge is likely to be of value for the conservation of this iconic fish species, by incorporating these size related differences in hypoxia tolerance in streams management. In light of the unusual oxyconforming response of inanga, a study examining the behavioural responses of this species to declining dissolved oxygen was performed. Inanga did not display a behaviour that might reduce energy expenditure during oxygen limitation; instead swimming activity and speed were elevated relative to normoxia. As hypoxia deepened inanga leaped out of the water, emersing themselves on a floating platform. Once emersed, fish exhibited an enhanced oxygen consumption rate compared to fish that remained in hypoxic water. Although this emersion behaviour was hypothesised to be of physiological advantage, both aquatic hypoxia and emersion resulted in similar physiological and biochemical consequences in inanga. While in hypoxic water oxygen availability seemed to be the limiting factor, in air failure of the circulatory system was hypothesised to be the cause of a similar metabolic signature to that found in aquatic hypoxia. Overall, inanga seemed to be not particularly well adapted to tolerate aquatic hypoxia. In light of the increasing likelihood of anthropogenic-induced hypoxia in inanga habitats, this is likely to have negative consequences for the future of inanga populations in the wild. Although this study provides the mechanisms behind the exceptional salinity tolerance of inanga, its susceptibility to hypoxia is likely to impose further constraints for the osmoregulatory processes that guarantee inanga survival during life cycle migrations. The results of the present study are relevant for understanding and managing the fishery of this economically- and culturally important fish species.

The syndrome known as Gastric Dilation Air Sacculitis (GDAS) has recently been described by Lumsden et al. (2002) for Chinook salmon (Oncorhynchus tshawytscha, Walbaum), in seawater (SW) culture in New Zealand. The syndrome is characterised by distended abdomens, gastric dilation and air sacculitis, increased feed conversion ratios (FCR) and mortality. Consequently, financial returns on affected stocks are greatly reduced. A study into the epidemiology and physiology of the syndrome was initiated, working with the major aquaculture company, The New Zealand King Salmon Company (NZKS). The study revealed causative factors of GDAS. GDAS was experimentally induced only in saltwater by feeding a commercially manufactured low-cohesion pelleted diet. Control groups were fed a different diet with high physical cohesion. Low-cohesion pellets have previously been associated with a high incidence of GDAS in commercial sea cages. These data implicated osmoregulatory stress and physical properties of the feed in GDAS development. In addition, gastrointestinal (GI) physiology in GDAS -affected and -control fish was characterised. The process of GDAS development in O. tshawytscha is characterised by a loss of smooth muscle tone of the stomach as it distends. Laplace's law (P= 2T/r, where P is the distending pressure, T is the tension in the wall and r is the radius of the cylinder) predicts that unless muscle mass increases, the ability of the stomach wall to contract will be lost and consequently a loss of GI motor function will result. Therefore, GI circular smooth muscle integrity in terms of (1) stimulated and maximal contractility, (2) osmoregulatory ability of the intestine and the (3) control of the GI system was studied in pathologically affected (+ve) and unaffected (-ve) smolt. Affected fish showed changes in GI circular smooth muscle function and osmoregulatory dysfunction. Feeding different diets induced distinct gastric evacuation patterns. The intestinal brake hypothesis is presented and argued to be the probable mechanism for GDAS development. GDAS (+ve) serum showed the presence of factors capable of contracting gut smooth muscle. In addition, potential humoral mediators of the intestinal brake in fish were investigated.

Thesis (Ph.D.)--University of Wyoming, 2008. / Title from PDF title page (viewed on August 9, 2009). Includes bibliographical references (p. 160-199).

Universiẗat, Diss., 2004--Köln.