Japanese eels (Anguilla japonicas) are snakelike fishes living in waters in the Asian region. In contrast to most fishes which are stenohalines that can only live in waters with a narrow range of salinity, Japanese eels are classified as euryhalines that can habitat in a broad range of salinity. As the lifecycle of Japanese eels consists of stages across fresh and seawater districts, a well-developed osmoregulation mechanism is needed to balance the intra- and extra- cellular osmolarity of the fishes throughout the seawater acclimation process. While fish gills are one of the organs that separating the ambient water and the inner body fluid of the fish, the fish gills of the Japanese eels have been studied as one of the most crucial organs for osmoregulation purposes. Yet, the osmoregulation and survival strategies of Japanese eels under hyperosmotic stress has not been fully elucidated. In chapter 2, this study has performed a transcriptome study on the ex vivo gill filament model of the Japanese eel to profile the molecular responses after a hypertonic treatment of 4 hours or 8 hours. The experiment is aimed to mimic the gill cells exposed to seawater in the seawater acclimation process of Japanese eels. A profile of differential expressed genes (DEGs) has been revealed that 577 DEGs were commonly upregulated and 711 DEGs were commonly downregulated in both 4- and 8-hours hypertonic treatment. Functional analysis and annotation have been processed with these DEGs, including Ingenuity Canonical Pathways analysis and gene ontology. These analyses have revealed that the cellular homeostasis of the gill cells has been disrupted and cell death responses has been induced by osmotic stress. The results have raises a concern that the maintenance of cellular viability and a cell death regulation mechanism are needed for the fishes to survive in the early stage of seawater acclimation. In chapter 3, this chapter demonstrated that gill cells in Japanese eels are susceptible to apoptosis when they are exposed to hyperosmotic treatments in both in vitro gill cell and the ex vivo gill filament model. To maintain the viability of the gills cells, two inhibitors of apoptosis, XIAP, and survivin, were seen to be expressed in gills cells. The expression of XIAP and survivin were upregulated by dexamethasone, which is an agonist mimicking the effect of cortisol on fishes in seawater acclimation. Meanwhile, the expression levels of the apoptosis executor, caspase 3, were downregulated. These data suggested that with the regulation of cortisol express in the fishes, XIAP and survivin are effective apoptosis regulators in the gill cells of Japanese eels. The study has demonstrated the molecular responses of the gills of Japanese eels exposed to hyperosmotic stress at the transcriptional level and post-translational level by using transcriptome studies and protein study respectively. The study has paved cell death regulation to be another the key field to study in understanding the ability of salinity tolerance in euryhalines.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18838号 / 理博第4096号 / 新制||理||1589(附属図書館) / 31789 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 阿形 清和, 教授 杤尾 豪人, 教授 森 和俊 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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Differences in osmoregulatory behaviors were studied in two stream-dwelling tree frogs (Pseudacris cadaverina and P. regilla) with different natural histories. This study supports the idea that the natural history of a species has a strong effect on behavior associated with osmoregulation.

In recent times, diminishing freshwater resources, due to the rapidly increasing drain of urban, industrial and agricultural activities in combination with the impact of climate change, has led to an urgent need to manage marine and brackish water environments more efficiently. Therefore the diversification of aquacultural practices, either by the introduction of new candidate species or by the adaptation of culture methods for existing species, is vital at a time when innovation and adaptability of the aquaculture industry is fundamental in order to maintain its sustainability. The Nile tilapia (Oreochromis niloticus, Linnaeus, 1758), which has now been spread well beyond its natural range, dominates tilapia aquaculture because of its adaptability and fast growth rate. Although not considered to be amongst the most salt tolerant of the cultured tilapia species, the Nile tilapia still offers considerable potential for culture in low-salinity water. An increase in knowledge of the limits and basis of salinity tolerance of Nile tilapia during the sensitive early life stages and the ability to predict responses of critical life-history stages to environmental change could prove invaluable in improving larval rearing techniques and extend the scope of this globally important fish species. The capability of early life stages of the Nile tilapia to withstand variations in salinity is due to their ability to osmoregulate, therefore the ontogeny of osmoregulation in the Nile tilapia was studied from spawning to yolk-sac absorption after exposure to different experimental conditions ranging from freshwater to 25 ppt. Eggs were able to withstand elevated rearing salinities up to 20 ppt, but transfer to 25 ppt induced 100% mortality by 48 h post-fertilisation. At all stages embryos and larvae hyper-regulated at lower salinities and hypo-regulated at higher salinities, relative to the salinity of the external media. Osmoregulatory capacity increased during development and from 2 days post-hatch onwards remained constant until yolk-sac absorption. Adjustments to larval osmolality, following abrupt transfer from freshwater to experimental salinities (12.5 and 20 ppt), appeared to follow a pattern of crisis and regulation, with whole-body osmolality for larvae stabilising at c. 48 h post-transfer for all treatments, regardless of age at time of transfer. Age at transfer to experimental salinities (7.5 – 20 ppt) had a significant positive effect on larval ability to osmoregulate; larvae transferred at 8 dph maintained a more constant range of whole body osmolality over the experimental salinities tested than larvae at hatch. Concomitantly, survival following transfer to experimental salinities increased with age. There was a significant effect (GLM; p < 0.05) of salinity of incubation and rearing media on the incidence of gross larval malformation that was seen to decline over the developmental period studied. It is well established that salinity exerts a strong influence on development and growth in early life stages of fishes therefore the effects of varying low salinities (0 - 25 ppt) on hatchability, survival, growth and energetic parameters were examined in the Nile tilapia during early life stages. Salinity up to 20 ppt was tolerable, although reduced hatching rates at 15 and 20 ppt suggest that these salinites may be less than optimal. Optimum timing of transfer of eggs from freshwater to elevated salinities was 3-4 h post-fertilisation, following manual stripping and fertilisation of eggs, however increasing incubation salinity lengthened the time taken to hatch. Salinity was related to dry body weight, with larvae in salinities greater than 15 ppt displaying, at hatch, a significantly (GLM: p < 0.05) lower body weight but containing greater yolk reserves than those in freshwater or lower salinities. Survival at yolk-sac absorption displayed a significant (GLM; p < 0.05) inverse relationship with increasing salinity and mortalities were particularly heavy in the higher salinities of 15, 20 and 25 ppt. Mortalities occurred primarily during early yolk-sac development yet stabilised from 5 dph onwards. Salinity had a negative effect on yolk absorption efficiency (YAE). Salinity-related differences in oxygen consumption rates were not detectable until 3 days post-hatch; oxygen consumption rates of larvae in freshwater between days 3 – 6 post-hatch were always significantly higher (GLM p < 0.05) than those in 7.5, 15, 20 and 25 ppt, however, on day 9 post-hatch this pattern was reversed and freshwater larvae had a significantly lower QO2 than those in elevated salinities. Salinity had a significant inverse effect on larval standard length, with elevated salinities producing shorter larvae from hatch until 6 dph, after which time there was no significant differences between treatments. Salinity had a significant effect on whole larval dry weight, with heavier larvae in elevated salinities throughout the yolk-sac period (GLM; p < 0.05). The ability of the Nile tilapia to withstand elevated salinity during early life stages is due to morphological and ultrastructural modifications of extrabranchial mitochondria-rich cells (MRCs) that confer an osmoregulatory capacity before the development of the adult osmoregulatory system. A clearly defined temporal staging of the appearance of these adaptive mechanisms, conferring ability to cope with varying environmental conditions during early development, was evident. Ontogenetic changes in MRC location, 2-dimensional surface area, density and general morphological changes were investigated in larvae incubated and reared in freshwater and brackish water (15 ppt) from hatch until yolk-sac absorption using Na+/K+-ATPase immunohistochemistry with a combination of microscope techniques. The pattern of MRC distribution was seen to change during development under both treatments, with cell density decreasing significantly on the body from hatch to 7 days post-hatch, but appearing on the inner opercular area at 3 days post-hatch and increasing significantly (GLM; p < 0.05) thereafter. Mitochondria-rich cells were always significantly (GLM; p < 0.05) denser in freshwater than in brackish water maintained larvae. In both freshwater and brackish water, MRCs located on the outer operculum and tail showed a marked increase in size with age, however, cells located on the abdominal epithelium of the yolk-sac and the inner operculum showed a significant decrease in size (GLM; p < 0.05) over time. Mitochondria-rich cells from brackish water maintained larvae from 1 day post-hatch onwards were always significantly larger (GLM; p < 0.05) than those maintained in freshwater. Preliminary scanning electron microscopy studies revealed structural differences in chloride cell morphology that varied according to environmental conditions. Mitochondria-rich cell morphology and function are intricately related and the plasticity or adaptive response of this cell to environmental changes is vital in preserving physiological homeostasis and contributes to fishes’ ability to inhabit diverse environments. Yolk-sac larvae were transferred from freshwater at 3 days post-hatch to 12.5 and 20 ppt and sampled at 24 and 48 h post-transfer. The use of scanning electron microscopy allowed a quantification of MRC, based on the appearance and surface area of their apical crypts, resulting in a reclassification of ‘sub-types’ i.e. Type I or absorptive, degenerating form (surface area range 5.2 – 19.6 μm2), Type II or active absorptive form (surface area range 1.1 – 15.7 μm2), Type III or differentiating form (surface area range 0.08 – 4.6 μm2) and Type IV or active secreting form (surface area range 4.1 – 11.7 μm2). In addition, the crypts of mucous cells were discriminated from those of MRCs based on the presence of globular extensions and similarly quantified.

Assignment (MPhil)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Many species of tilapia such as Oreochromis mossambicus are euryhaline, able to adapt to different salinity waters. Their ability to withstand high salinity levels has given rise to the possibility of using tilapia as baitfish for tuna line fishery. The purpose of the study was to determine the survival rate of tilapia O. mossambicus during direct transfer from freshwater to the salinity levels of 0, 15, 20, 22.5, 25, 27.5, 30, 32.5, and 35 ppt. The data was analysed through means of univariate ANOVAand regression analysis. O. mossambicus showed no mortality to all salinity regimes up to 25 ppt. Mortality was observed at 27.5 ppt, with 100% mortality at 35 ppt. LC 50 and LC 90 were found to be 30.5 and 34.2 ppt, respectively. The results indicate that tilapia (0. mossambicus) will survive a direct transfer to salinities up to 25 ppt. acclimation will be required in the event of transfer to salinity levels above 25 ppt, in order to prevent significant levels of mortalities. / AFRIKAANSE OPSOMMING: Meeste van die tilapia spesies soos Oreochremis mossambicus het die vermoë om by water van verskillende soutgehaltes aantepas. Dit is hierdie vermoë om hoë sout vlakke te weerstaan wat die moontlikheid vir gebruik as lewende aas in die tuna langlyn visvangbedryf moontlik maak. Die doel van hierdie studie was om die oorlewingsvlak van tilapia, O. mossambicus te bepaal by die oorplasing van varswater direk na soutwater by vlakke van 0, 15, 20, 22.5, 25, 27.5, 30, 32.5, en 35 dele per duisend. Die data is verwerk deur gebruik te maak van eenvariant ANOVAen regressie analises. O. mossambicus het geen mortaliteite tot gevolg gehad by al die oorplasings van vlakke tot en met 25 dele per duisend sout nie. Mortaliteite is wel gevind vanaf 27.5 dele per duisend, met 100 % mortaliteite by 35 dele per duisend. LC 50 en LC90 was gewees 30.5 en 34.2 dele per duisend onderskeidelik. Die resultate toon aan dat tilapia (0. mossambicus) sal oorleef by direkte oorplasing na soutwater by vlakke van tot en met 25 dele per duisend. Tilapia wat na hoër vlakke as 25 dele per duisend oorgeplaas wil word, sal eers geleidelik moet akklimatiseer om mortaliteite te beperk.

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.