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京都大学 / 新制・課程博士 / 博士(理学) / 甲第24456号 / 理博第4955号 / 新制||理||1707(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 渡辺 勝敏, 教授 曽田 貞滋, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Aquatic biodiversity continues to decline as humans modify the landscape. A population-level approach is necessary to address the mechanisms of impairment in urban stream habitats. When estimating population-level parameters, incomplete detection of individuals must be accounted for to ensure unbiased estimates. In this thesis, I examined differences in the detection probabilities of stream fishes, and used estimates of size/age-specific detection probabilities to reduce bias in estimates of the reproductive success of various fish species in urban and forested stream habitats. In Chapter 1, I examined differences in detection probabilities of stream fishes among electrofishing passes and size/age groups in the middle New River basin, Virginia. I also examined differences in detection probabilities between two physiographic regions: the middle New River basin, and the upper Wabash River basin, Indiana; and evaluated differences between single- and multiple-season estimation methods. I found that for most species, detection probabilities do not differ among electrofishing passes, size/age classes, between the two regions, or between single- and multiple-season estimation methods. I used size/age-specific estimates of detection probabilities to remove bias from relative abundance estimates of steam fish populations in Chapter 2. In Chapter 2, I examined the reproductive success of six lithophilic and speleophilic fishes in urban and forested reaches of 2nd-4th Strahler-order streams in the middle New River basin. I found that binary classification is a poor method of quantifying reproductive success, that the age distributions of many lithophilic and speleophilic species are dominated by adult individuals in urban habitats, and that the population growth rates of speleophils is reduced in urban habitats. These results suggest that although detection probabilities may be equal among various sources of variation, managers should verify this assumption before assuming equal detectability. The results also suggest that reduced reproductive success of speleophilic and lithophilic species in urban stream habitats may be a mechanism of their impairment. / Master of Science

To understand the ‘osmorespiratory compromise’ (the trade-off in gill function between ion and respiratory gas exchange) during hypoxia in freshwater fish, a species-specific approach was utilized where general ionoregulatory responses to hypoxia were compared in rainbow trout (Oncorhynchus mykiss, a hypoxia-intolerant freshwater fish), and in two hypoxia-tolerant species (the goldfish Carassius auratus and the Amazonian oscar Astronotus ocellatus). In the latter two species, the dual stress situation of hypoxia plus feeding was also explored. Measurements included unidirectional and net Na^+ flux rates, ammonia excretion rates, net K^+ loss rates, branchial Na^+/K^+- ATPase and H^+- ATPase activities, and branchial morphology by scanning electron microscopy (trout and oscar only). In trout, environmental hypoxia induced complex changes in gill ionoregulatory function, where the direction and magnitude varied with both the extent and duration of the hypoxia regime. The changes in ion-regulation observed in trout in response to hypoxia indicated that the osmorespiratory compromise in this hypoxia-intolerant species was different and more complex compared to its manifestation in oscar and goldfish. This could be attributed to the adaptive physiology of the trout to oxygen-rich environments and its intolerance to low environmental oxygen availability. In both of the hypoxia-tolerant species (oscar and goldfish), there was a general reduction in gill permeability in response to severe hypoxia regardless of feeding regime, rather different from the complex patterns seen in the hypoxia-intolerant trout. However, the effects of feeding on this phenomenon differed between these species. Fed goldfish had elevated branchial fluxes that were effectively turned down during hypoxia compared to baseline flux rates maintained by starved goldfish. In contrast, fed oscars had lower fluxes compared to starved fish. Although both fed and starved fish suppressed their branchial fluxes with severe hypoxia, fed oscars delayed the turning down of fluxes. Overall, our results indicate that feeding exerts opposite effects on gill ionoregulatory function in these two hypoxia-tolerant species, and thereby differentially modulates the responses to hypoxia. These differences may relate to differences in water chemistry. Furthermore, the manifestation of the osmorespiratory compromise during hypoxia appears to be rather different from the phenomenon during exercise. / Thesis / Master of Science (MSc)

Animal hosts provide associated microorganisms with suitable ecological niches in their intestines. Microbes help their hosts to digest food, protect against pathogens, and influence the host’s metabolisms. Compositional variation of gut microbial communities is common among hosts, and may affect the health status of hosts. Diet and genetic factors are well known to influence the assembly of gut microbial communities. This thesis focuses on disentangling the contributions of factors including host genetics (sex), diet, environment, and other ecological processes to the assembly of gut microbial communities in freshwater fish. The association between gut microbial communities and fish condition is also evaluated in this thesis. Applying metacommunity theory, we found environmental factors including fish habitat, fish species, their diet, dispersal factors including microbes from fish diet, and ecological drift contributed to the assembly of fish gut microbial communities. The proportion of their contribution varied between fish species, where ecological drift explained more in perch than in roach. Under natural conditions fish populations face the risk of predation, which can induce competition and impose predation stress within prey individuals. This can therefore lead to changes in their diet qualities and quantities. In this thesis, it was shown that fish diet in terms of qualities and quantities significantly influenced the overall gut microbial composition, and this influence was dependent on fish sex, a host genetic factor. Predation stress was also suggested to significantly decrease the species richness. Furthermore, when fish were experiencing a diet shift, we showed that different bacterial phyla from novel food had different colonization success in the intestine, and this colonization success was positively influenced by predation stress. Fish condition was suggested in this thesis to be affected by gut microbial composition, especially by the contributions of the bacterial phyla Tenericutes and Actinobacteria.

O presente trabalho teve como objetivos: (1) avaliar o rendimento e as características físico-químicas dos óleos de vísceras de truta arco-íris (Oncorhynchus mykiss), pacu (Piaractus mesopotamicus) e curimbatá (Prochilodus spp.) extraídos por congelamento lento, como mecanismo para liberar os lipídios do material bruto, e (2) comparar as etapas do refinamento químico (degomagem, neutralização, lavagem, secagem e branqueamento) de dois óleos de truta arco-íris, extraídos por congelamento lento e por um processo termo-mecânico. As matérias primas foram coletadas em diferentes municípios dos Estados de São Paulo e Paraná. Foram determinados os rendimentos, teor de ácidos graxos livres, índice de peróxidos, índice de iodo, índice de saponificação, densidade, índice de refração, viscosidade e perfil de ácidos graxos nos óleos brutos (truta arco-íris, pacu e curimbatá) e nos óleos submetidos a processos de refinamento (truta arco-íris). Os rendimentos de óleo de vísceras de truta arco-íris, pacu e curimbatá, foram de 27,58%, 42,53 e 13,75% respectivamente. Os parâmetros físicoquímicos avaliados em todos os óleos (óleos brutos das vísceras das três espécies de peixes e óleos de truta arco-íris submetidos a refinamento) encontraram-se dentro dos níveis referenciados para óleos de peixe brutos e refinados. O perfil de ácidos graxos foi variável entre os óleos brutos das três espécies, sendo que o óleo de curimbatá apresentou maiores níveis dos ácidos eicosapentaenoico, docosaexaenoico e araquidônico, e menor relação n-6/n-3, portanto de melhor qualidade nutricional, provavelmente por ter sido capturado na natureza. Entretanto, devido à alta disponibilidade de truta arco-íris, considera-se mais viável a produção de complementos nutricionais de ácidos graxos poliinsaturados da família n-3 nesta espécie, desde que o perfil lipídico do óleo esteja adequado. Os teores de ácidos graxos da família n-3, dos óleos de truta arco-íris foram menores que os relatados na literatura; podendo ser influenciados pela dieta dos animais. O tipo de extração produziu diferenças no perfil de ácidos graxos tanto nos óleos brutos quanto nos óleos branqueados. Destaca-se maior teor de ácidos graxos com configuração trans no óleo (bruto e branqueado) de truta arco-íris, extraído por autoclavagem devido provavelmente à utilização de altas temperaturas durante a extração. A técnica de extração por congelamento foi eficiente em termos de rendimento e qualidade dos óleos extraídos das vísceras dos peixes avaliados, e possivelmente seja eficiente também em outras espécies e matérias primas. / This study aimed to: (1) evaluate the performance and physicochemical characteristics of viscera oils from rainbow trout (Oncorhynchus mykiss), pacu (Piaractus mesopotamicus) and curimbatá (Prochilodus spp.) extracted by slow freezing, as a mechanism to release the lipids of the crude material, and (2) compare the stages of chemical refining (degumming, neutralization, washing, drying and bleaching) of two rainbow trout oils, extracted by slow freezing and by a thermo-mechanic process. The raw materials were collected in different places of the States of São Paulo and Parana. Viscera oil yields were determined, content of free fatty acids, peroxide value, iodine value, saponification value, density, refractive index, viscosity and fatty acid profile of the crude oils (rainbow trout, pacu and curimbatá) and of the rainbow trout oils underwent the refinement process. The oil yields of viscera rainbow trout, pacu and curimbatá, were 27.58%, 42.53 and 13.75% respectively. The physico-chemical parameters evaluated in all the oils (crude oils from the viscera of the three species of fish and rainbow trout oils subjected to refinement) were within the referenced levels for the crude and refined fish oils. The fatty acid profile varied between the crude oils from the three species. Curimbatá viscera oil, showed higher levels of eicosapentaenoic acid, docosahexaenoic and arachidonic acid, and lower n-6/n-3 ratio therefore of higher nutritional quality, probably for having been caught in the wild. However, due to the high availability of rainbow trout, it is more viable the production of nutritional supplements of n-3 polyunsaturated fatty acids with by-products from this species, since their oil lipid profile is suitable. The levels of n-3 fatty acids of rainbow trout oils were lower than those reported in the literaturepossibly influenced by the diet. The type of extraction produced differences in the fatty acid profile both in crude oils and bleached oils. It stands out higher content of fatty acids with trans configuration in the rainbow trout oil (crude and bleached), obtained by autoclaving probably due to the use of high temperatures during extraction. The freeze-extraction technique was efficient in terms of yield and quality of oils extracted from the viscera of fishes evaluated and could also be effective in other species and raw materials.

The arrival of a non-native species to has the potential to shape native communities by influencing ecological interactions such as predation, foraging, competition and disease transfer. A designation of invasive is applied to an introduced non-native species that has the potential to threaten the continued wellbeing of a native species, pose a risk to human health or negatively impact the economy. The European bullhead (Cottus perifretum) is a freshwater benthic-dwelling fish that is native to England but considered invasive in Scotland. The species was first reported in Scotland in the 1950's and thriving populations are now established in the waters of the Clyde, Forth and Tweed catchments. Bullhead presence is thought to negatively impact native stone loach (Barbatula barbatula) and brown trout (Salmo trutta) parr, due to shared preferences for habitat and prey resources. They are also thought to prey upon the eggs of native Atlantic salmon (Salmo salar) and brown trout, two species that are of high commercial value in Scotland. In other areas of introduction, bullheads have been found to increase parasite infection rates in native fishes. The species therefore has the capacity to incite competition and alter parasite/host interactions in areas of introduction, to the potential detriment of native fauna and the Scottish economy. The European bullhead has been the subject of considerable taxonomic scrutiny in recent years, resulting in its reclassification as a species complex. What was once considered a single species with a distribution encompassing Europe, Russia, Asia and Scandinavia, has been shown to consist of at least 15 distinct species. Genetic examination of bullheads from England confirmed the presence of Cottus perifretum, not Cottus gobio as traditionally listed in all UK literature and legislation. Native English bullhead is currently protected under Annex II of the European Commission Habitats Directive 92/43/EEC, based on the historic assumption that the species present is C. gobio. Analysis of the taxonomic identity of Scottish bullheads has remained outstanding. In this study the invasive status of bullhead was explored by examining feeding and parasitological interactions between bullheads and native freshwater communities in south-east Scotland. An assessment of the feeding preferences of native brown trout and stone loach in the presence and absence of bullheads tested competition for prey resources. Parasitological interactions were investigated by examining the shared parasite fauna of bullheads and native fish and invertebrate species. Bullheads from the Clyde and Forth catchments were analysed to provide a molecular and morphological description of this introduced species. Phylogenetic analysis of COX1 sequences obtained from Scottish bullheads, and a pair-wise distance calculation based on a Kimura 2-parameter model, showed that samples clustered in a distinct clade with English C. perifretum. Significant intraspecific variation was reported in all morphological features examined, but pooled data also revealed a resemblance to the published description provided for C. perifretum. Scottish bullhead is therefore confirmed to be an introduced pocket of the native English species, which is considered under threat in some areas due to habitat modifications and population decline. Comparisons between the dietary compositions of bullheads, brown trout and stone loach showed that the prey selection of brown trout and stone loach varied in the sample locations that contained co-occuring bullheads, when compared to locations where bullheads were absent. However, no direct evidence of trophic competition between bullheads and either brown trout or stone loach was reported. An examination of parasitological interactions recovered eight parasite species from four distinct taxonomic groups in total, of which four species (Echinorhynchus truttae, Apatemon gracilis, Diplostomum volvens and Raphidascaris acus) were shared between bullheads and one or more native fishes. Echinorhynchus truttae was also shared with gammarid crustaceans. Bullhead presence was found to coincide with D. volvens infections in European minnow (Phoxinus phoxinus) and a reduced prevalence of E. truttae in brown trout. Diplostomum volvens was only reported from minnow found in sites with bullheads, strongly suggesting bullheads were responsible for introducing this parasite to native minnows. Bullheads functioned as an alternative host for E. truttae, diluting brown trout parasite loads and reducing overall infection rates in sites where bullhead and brown trout co-exist. The findings reported for both feeding preference and parasite burdens in the presence and absence of bullheads suggest that bullheads do have some effect on the ecology of native species, but these are considered minimal and unlikely to impact the long-term survival of native species. Eradication of Scottish bullhead may contradict the conservation effort that is currently in place. Given the current lack of evidence to validate bullhead's invasive qualities and the recent confirmation of its genetic lineage, revisiting the designation of the bullhead as invasive is warranted. Active eradication should be treated with caution until a significant negative impact can be proven.

Orientador: Claudio de Oliveira / Resumo: Embora a ictiofauna Neotropical seja considerada a mais diversa do mundo, apenas 12,2% das espécies de peixes tiveram cariótipos descritos. A dificuldade de se obter preparações cromossômicas de boa qualidade para algumas espécies de peixes Neotropicais ainda é um dos grandes desafios na área de Citogenética, principalmente para peixes de pequeno e grande porte. Uma alternativa é uso de metodologias indiretas, tais como cultura de células, amplamente utilizada e estabelecida em mamíferos, entretanto para peixes ainda é pouco empregada pela dificuldade de padronização da técnica de isolamento e manutenção das culturas celulares. Dessa forma, o presente estudo teve como objetivo o desenvolvimento de uma metodologia de cultura de células aderentes para obtenção de cromossomos mitóticos em peixes neotropicais, estabelecendo linhagens celulares de diferentes espécies e a determinação de suas características citogenéticas. Seis espécies de peixes Neotropicais da ordem Characiformes foram utilizadas para obtenção das culturas primárias, desenvolvimento e criopreservação das linhagens celulares e analise citogeneticamente. Para obtenção das linhagens, células de tecido muscular foram isoladas usando enzimas proteolíticas. As células isoladas foram cultivadas em frascos ou placas tratadas com um filme à base de gelatina e mantidas à 27,5ºC, 5% CO2 em meio completo (DMEM+soro fetal bovino+antibióticos+antimicóticos). Quando as células cobriram toda a superfície do substrato, elas foram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Although Neotropical ichthyofauna is considered the most diverse in the world, only 12.2% had karyotypes described. The difficulty of obtaining good quality chromosome preparations for some Neotropical fish species is still one of the major challenges in the area of cytogenetics, especially for small and large fish. An alternative is the use of indirect methodologies, such as cell culture, widely used and established in mammals, however for fish is still little used for the difficulty of standardization of the technique of isolation and maintenance of cell culture. Thus, the present study aimed to develop a methodology for adherent cell culture to obtain mitotic chromosomes in Neotropical fish, establishmenting cell lines of different species and the cytogenetic characteristics of these lines. Six species of Neotropical fish of the order Characiformes were used to obtain primary cell, development and cryopreservation of the cell lines and cytogenetic analysis. To obtain the cell lines, muscle tissue were isolated using proteolytic enzymes. The isolated cells were cultured in flasks or plates treated with a gelatin-based film and maintained at 27.5 °C, 5% CO2 in complete medium (DMEM + fetal bovine serum + antibiotics + antimycotics). When the cells covered almost all surface of the substrate, they were transferred to new flasks to obtain chromosome preparations and cryopreservation. The chromosome preparations of the cell lines were performed in different passages in the spec... (Complete abstract click electronic access below) / Doutor

O presente trabalho teve como objetivos: (1) avaliar o rendimento e as características físico-químicas dos óleos de vísceras de truta arco-íris (Oncorhynchus mykiss), pacu (Piaractus mesopotamicus) e curimbatá (Prochilodus spp.) extraídos por congelamento lento, como mecanismo para liberar os lipídios do material bruto, e (2) comparar as etapas do refinamento químico (degomagem, neutralização, lavagem, secagem e branqueamento) de dois óleos de truta arco-íris, extraídos por congelamento lento e por um processo termo-mecânico. As matérias primas foram coletadas em diferentes municípios dos Estados de São Paulo e Paraná. Foram determinados os rendimentos, teor de ácidos graxos livres, índice de peróxidos, índice de iodo, índice de saponificação, densidade, índice de refração, viscosidade e perfil de ácidos graxos nos óleos brutos (truta arco-íris, pacu e curimbatá) e nos óleos submetidos a processos de refinamento (truta arco-íris). Os rendimentos de óleo de vísceras de truta arco-íris, pacu e curimbatá, foram de 27,58%, 42,53 e 13,75% respectivamente. Os parâmetros físicoquímicos avaliados em todos os óleos (óleos brutos das vísceras das três espécies de peixes e óleos de truta arco-íris submetidos a refinamento) encontraram-se dentro dos níveis referenciados para óleos de peixe brutos e refinados. O perfil de ácidos graxos foi variável entre os óleos brutos das três espécies, sendo que o óleo de curimbatá apresentou maiores níveis dos ácidos eicosapentaenoico, docosaexaenoico e araquidônico, e menor relação n-6/n-3, portanto de melhor qualidade nutricional, provavelmente por ter sido capturado na natureza. Entretanto, devido à alta disponibilidade de truta arco-íris, considera-se mais viável a produção de complementos nutricionais de ácidos graxos poliinsaturados da família n-3 nesta espécie, desde que o perfil lipídico do óleo esteja adequado. Os teores de ácidos graxos da família n-3, dos óleos de truta arco-íris foram menores que os relatados na literatura; podendo ser influenciados pela dieta dos animais. O tipo de extração produziu diferenças no perfil de ácidos graxos tanto nos óleos brutos quanto nos óleos branqueados. Destaca-se maior teor de ácidos graxos com configuração trans no óleo (bruto e branqueado) de truta arco-íris, extraído por autoclavagem devido provavelmente à utilização de altas temperaturas durante a extração. A técnica de extração por congelamento foi eficiente em termos de rendimento e qualidade dos óleos extraídos das vísceras dos peixes avaliados, e possivelmente seja eficiente também em outras espécies e matérias primas. / This study aimed to: (1) evaluate the performance and physicochemical characteristics of viscera oils from rainbow trout (Oncorhynchus mykiss), pacu (Piaractus mesopotamicus) and curimbatá (Prochilodus spp.) extracted by slow freezing, as a mechanism to release the lipids of the crude material, and (2) compare the stages of chemical refining (degumming, neutralization, washing, drying and bleaching) of two rainbow trout oils, extracted by slow freezing and by a thermo-mechanic process. The raw materials were collected in different places of the States of São Paulo and Parana. Viscera oil yields were determined, content of free fatty acids, peroxide value, iodine value, saponification value, density, refractive index, viscosity and fatty acid profile of the crude oils (rainbow trout, pacu and curimbatá) and of the rainbow trout oils underwent the refinement process. The oil yields of viscera rainbow trout, pacu and curimbatá, were 27.58%, 42.53 and 13.75% respectively. The physico-chemical parameters evaluated in all the oils (crude oils from the viscera of the three species of fish and rainbow trout oils subjected to refinement) were within the referenced levels for the crude and refined fish oils. The fatty acid profile varied between the crude oils from the three species. Curimbatá viscera oil, showed higher levels of eicosapentaenoic acid, docosahexaenoic and arachidonic acid, and lower n-6/n-3 ratio therefore of higher nutritional quality, probably for having been caught in the wild. However, due to the high availability of rainbow trout, it is more viable the production of nutritional supplements of n-3 polyunsaturated fatty acids with by-products from this species, since their oil lipid profile is suitable. The levels of n-3 fatty acids of rainbow trout oils were lower than those reported in the literaturepossibly influenced by the diet. The type of extraction produced differences in the fatty acid profile both in crude oils and bleached oils. It stands out higher content of fatty acids with trans configuration in the rainbow trout oil (crude and bleached), obtained by autoclaving probably due to the use of high temperatures during extraction. The freeze-extraction technique was efficient in terms of yield and quality of oils extracted from the viscera of fishes evaluated and could also be effective in other species and raw materials.

Thesis (M.Sc. (Physiology)) -- University of Limpopo, 1993. / Refer to the document / University of Limpopo Research Office