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Bei zwei Fließgewässern (Lockwitzbach und Körsch) wurde ein Expositions- und Effektmonitoring an Probenahmestellen jeweils ober- und unterhalb des Einleiters eines kommunalen Klärwerks über einen Zeitraum von zwei Jahren durchgeführt. Gegenstand der vorliegenden Arbeit waren dabei die Untersuchungen zur Populationsstruktur und -dynamik von Gammarus fossarum (Amphipoda, Crustacea). Folgende Populationsvariablen wurden erfasst: Abundanz, Geschlechterverhältnis, Anteil von Juvenilen, Anteil brütender Weibchen, Fekundität, Körperlängen und Infektion mit Acanthocephalen. Erstmalig wurde Intersexualität bei G. fossarum festgestellt. Im Lockwitzbach war der Anteil an Intersexen bei den adulten Gammariden mit etwa 5 - 15 % höher als in der Körsch mit < 1 %. Intersexualität wurde nicht durch die Einleiter induziert. Bei den Intersexen handelt es sich um funktionelle Weibchen. Wodurch dieses Phänomen ausgelöst wird und welche Bedeutung es für die Population hat, ist unbekannt. In den Freilanduntersuchungen wurden im Projekt Xehogamm (Umweltbundesamt Berlin, FKZ 299 65 221/05), in dessen Rahmen diese Dissertation entstand, verschiedene Umweltchemikalien im Bachwasser analysiert. Bei Vertebraten ist eine östrogene Wirksamkeit dieser Substanzen bekannt oder wird vermutet. In den Gammaridenpopulationen unterhalb der Klärwerkseinleiter waren wichtige populationsrelevante Variablen im Vergleich zu oberhalb verändert. An der unteren Probenahmestelle bei der Körsch war der Anteil der Juvenilen mit der kleinsten Körperlänge sowie der Anteil brütender Weibchen erniedrigt. Tendenziell traf dies auch für die untere Probenahmestelle beim Lockwitzbach zu. Bei der Körsch ist außerdem der frühere Beginn der herbstlichen Reproduktionspause und die geringere Körperlänge adulter Gammariden möglicherweise auf endokrine Umweltchemikalien im Wasser des Einleiters zurückzuführen. Von den nachgewiesenen Substanzen wurde eine Einzelsubstanz, Bisphenol A, für ein Fließrinnenexperiment ausgewählt. In künstlichen Fließrinnen im Gewächshaus wurde G. fossarum paarweise, in Gruppen sowie als größenstrukturierte Population in der Fließrinne selbst über 103 Tage exponiert, und verschiedene Populationsvariablen wurden erfasst. Die Nominalkonzentrationen von Bisphenol A in drei Fließrinnen betrugen 5, 50 und 500 µg/L. In den ersten drei aufeinanderfolgenden Bruten zeigte sich in der höchsten Bisphenol A-Konzentration die größte Brutgröße. Im weiteren Verlauf des Experiments übte Bisphenol A einen hemmenden Einfluss auf die Reproduktion der Gammariden aus. Basierend auf den Nominalkonzentrationen wurden folgende EC10-Werte berechnet: Anteil reproduzierender Weibchen für die 4. Brut (bei Gammaridenpaaren): 22 µg/L, Brutgröße der 4. Brut (bei Gammaridenpaaren): 11 µg/L, Anteil brütender Weibchen (Population): 212 µg/L und Anteil Juveniler (Population): 153 µg/L Bisphenol A. Die EC10-Werte basierend auf Effektivkonzentrationen für dieselben Endpunkte betragen: bei den Paaren 1,1 µg/L (reproduzierende Weibchen) und 0,5 µg/L (Brutgröße), in der Population 10 µg/L (Anteil brütender Weibchen und Anteil Juveniler). Im Fließrinnenexperiment wurde Bisphenol A zwar in höheren Konzentrationen als im Freiland vorhanden eingesetzt, die beobachteten Effekte weisen jedoch in dieselbe Richtung wie die Effekte im Freiland. Daher kann nicht ausgeschlossen werden, dass auch die Effekte im Freiland auf einer endokrinen Wirkung von Umweltchemikalien beruhen könnten. Zusätzlich wurde der Einfluss von Bisphenol A auf die Osmoregulation bei G. lacustris untersucht, wobei unterschiedliche Effekte auf die Natrium- und Calciumkonzentration, nicht jedoch auf die Osmolalität, in der Hämolymphe nachgewiesen wurden. Anlage: Rohdaten (3,87 MB)- Nutzung: Referat Informationsvermittlung der SLUB

Bei zwei Fließgewässern (Lockwitzbach und Körsch) wurde ein Expositions- und Effektmonitoring an Probenahmestellen jeweils ober- und unterhalb des Einleiters eines kommunalen Klärwerks über einen Zeitraum von zwei Jahren durchgeführt. Gegenstand der vorliegenden Arbeit waren dabei die Untersuchungen zur Populationsstruktur und -dynamik von Gammarus fossarum (Amphipoda, Crustacea). Folgende Populationsvariablen wurden erfasst: Abundanz, Geschlechterverhältnis, Anteil von Juvenilen, Anteil brütender Weibchen, Fekundität, Körperlängen und Infektion mit Acanthocephalen. Erstmalig wurde Intersexualität bei G. fossarum festgestellt. Im Lockwitzbach war der Anteil an Intersexen bei den adulten Gammariden mit etwa 5 - 15 % höher als in der Körsch mit < 1 %. Intersexualität wurde nicht durch die Einleiter induziert. Bei den Intersexen handelt es sich um funktionelle Weibchen. Wodurch dieses Phänomen ausgelöst wird und welche Bedeutung es für die Population hat, ist unbekannt. In den Freilanduntersuchungen wurden im Projekt Xehogamm (Umweltbundesamt Berlin, FKZ 299 65 221/05), in dessen Rahmen diese Dissertation entstand, verschiedene Umweltchemikalien im Bachwasser analysiert. Bei Vertebraten ist eine östrogene Wirksamkeit dieser Substanzen bekannt oder wird vermutet. In den Gammaridenpopulationen unterhalb der Klärwerkseinleiter waren wichtige populationsrelevante Variablen im Vergleich zu oberhalb verändert. An der unteren Probenahmestelle bei der Körsch war der Anteil der Juvenilen mit der kleinsten Körperlänge sowie der Anteil brütender Weibchen erniedrigt. Tendenziell traf dies auch für die untere Probenahmestelle beim Lockwitzbach zu. Bei der Körsch ist außerdem der frühere Beginn der herbstlichen Reproduktionspause und die geringere Körperlänge adulter Gammariden möglicherweise auf endokrine Umweltchemikalien im Wasser des Einleiters zurückzuführen. Von den nachgewiesenen Substanzen wurde eine Einzelsubstanz, Bisphenol A, für ein Fließrinnenexperiment ausgewählt. In künstlichen Fließrinnen im Gewächshaus wurde G. fossarum paarweise, in Gruppen sowie als größenstrukturierte Population in der Fließrinne selbst über 103 Tage exponiert, und verschiedene Populationsvariablen wurden erfasst. Die Nominalkonzentrationen von Bisphenol A in drei Fließrinnen betrugen 5, 50 und 500 µg/L. In den ersten drei aufeinanderfolgenden Bruten zeigte sich in der höchsten Bisphenol A-Konzentration die größte Brutgröße. Im weiteren Verlauf des Experiments übte Bisphenol A einen hemmenden Einfluss auf die Reproduktion der Gammariden aus. Basierend auf den Nominalkonzentrationen wurden folgende EC10-Werte berechnet: Anteil reproduzierender Weibchen für die 4. Brut (bei Gammaridenpaaren): 22 µg/L, Brutgröße der 4. Brut (bei Gammaridenpaaren): 11 µg/L, Anteil brütender Weibchen (Population): 212 µg/L und Anteil Juveniler (Population): 153 µg/L Bisphenol A. Die EC10-Werte basierend auf Effektivkonzentrationen für dieselben Endpunkte betragen: bei den Paaren 1,1 µg/L (reproduzierende Weibchen) und 0,5 µg/L (Brutgröße), in der Population 10 µg/L (Anteil brütender Weibchen und Anteil Juveniler). Im Fließrinnenexperiment wurde Bisphenol A zwar in höheren Konzentrationen als im Freiland vorhanden eingesetzt, die beobachteten Effekte weisen jedoch in dieselbe Richtung wie die Effekte im Freiland. Daher kann nicht ausgeschlossen werden, dass auch die Effekte im Freiland auf einer endokrinen Wirkung von Umweltchemikalien beruhen könnten. Zusätzlich wurde der Einfluss von Bisphenol A auf die Osmoregulation bei G. lacustris untersucht, wobei unterschiedliche Effekte auf die Natrium- und Calciumkonzentration, nicht jedoch auf die Osmolalität, in der Hämolymphe nachgewiesen wurden. Anlage: Rohdaten (3,87 MB)- Nutzung: Referat Informationsvermittlung der SLUB

Osmoregulation is an essential process to maintain water and ionic balance and when euryhaline fish move between freshwater and seawater environments as part of their life cycle this presents additional osmoregulatory challenges. Migrating fish can be exposed in both environments to pollutants such as endocrine disrupting chemicals (EDCs) that include natural hormones (e.g. 17β-estradiol; E2), synthetic hormones (e.g. 17α-ethinylestradiol; EE2), and industrial chemicals (e.g. nonylphenol). The focus of this thesis was to study the effects of different categories of EDCs on the osmoregulatory functions of euryhaline fish such as three-spined sticklebacks (Gasterosteus aculeatus) and rainbow trout (Oncorhynchus mykiss). Osmoregulatory variables (such as osmolality, water and ionic content) were compared in plasma and tissues (white muscle and carcass) of rainbow trout. This validated the use of specific tissue parameters as a surrogate of plasma responses to various osmoregulatory challenges. Waterborne exposure to 17α-ethinylestradiol revealed differential sensitivity of vitellogenesis in the three-spined sticklebacks (no induction) and rainbow trout, but had a significant effect on calcium homeostasis in both species. Intraperitoneal implants of 17β-estradiol reduced CaCO3 production and apparent water absorption in the intestine and increased in tissue calcium stores of seawater-acclimated trout, but fish were able to compensate and showed no overall osmoregulatory disturbance. Waterborne exposure to nonylphenol in freshwater trout was also investigated, but no effects on osmoregulation were found up to 2 ng/l. Overall, estrogens can affect osmoregulation differentially in euryhaline fish species, and sometimes at EDC levels lower than the threshold for reproductive effects (i.e. vitellogenin induction).

This thesis deals with the Biology Olympiad and remaking of selected tasks of region round. The remaked tasks can be used in highschool education. The thesis includes theoretical and practical part. The theoretical part provides general information about the Biology Olympiad (BiO), charts the achievements of Czech students in International Biology Olympiad (IBO), includes literary review of selected topics. The practical part encompass four remaked tasks of different topics: Eucaryotic cell as a mosaic, Water in life of animals, Animals' areas and their distribution, History of the flora in Czech Republic. The tasks are processed in the form of worksheets. The worksheets were tested at schools. Knowledge of students was tested by using of created pretests and posttests. The practical part also presents the result of research based on a questionnaire survey focused on the involvement of school in BiO and teachers' opinions on these topics.

Como resultado do seu passado evolutivo, a família dos camarões Palaemonidae reúne representantes de ambientes osmóticos dos mais variados. Sejam de ambientes marinhos, estuarinos ou dulcícolas, estáveis ou variáveis, as espécies destes camarões mantêm a concentração osmo-iônica da hemolinfa independente da concentração do meio. Essas espécies hiper-regulam a osmolalidade e íons da hemolinfa em meio diluído e água doce e hipo-regulam em meio concentrado ou água do mar. Um importante local de transporte iônico envolvido na regulação osmo-iônica é o epitélio brânquial, pois nas membranas de seus ionócitos constituintes encontra-se um conjunto de transportadores que efetuam o movimento transepitelial de íons. Dentre estes transportadores, o simportador Na+, K+, 2Cl- (NKCC) é considerado ter um papel na secreção de sal, objetivo primário dessa investigação. Espécies representativas de habitat marinho do gênero Palaemon foram coletadas em regiões de estuário e de poça de maré, e de habitat dulcícola do gênero Macrobrachium foram coletadas em rios que desembocam no mar e também em riachos continentais sem influência do aporte salobro. Avaliou-se os limites letais de salinidade superior (LSS50) das espécies marinhas P. northropi e P. pandaliformis e dulcícolas diádromas M. acanthurus, M. olfersi, M. amazonicum que dependem de água salobra para completo desenvolvimento larval, e hololimnéticas M. potiuna e M. brasiliense com ciclo reprodutivo completo em água doce. Objetivou-se aqui (i) caracterizar os mecanismos de hiperregulação (condição controle de 18 S P. northropi, 17 S P. pandaliformis, água doce <0,5 S nas espécies de Macrobrachium) e hiporegulação [a curto (24 h) e longo prazo (120 h) em salinidade correspondente a 80% da LSS50] da osmolalidade e [Cl-] da hemolinfa, da expressão gênica e proteica e a localização por imunofluorescência do NKCC nos ionócitos branquiais; (ii) da existência de um padrão filogenético nesses parâmetros; e (iii) testar as hipóteses de um efeito da salinidade na evolução da expressão gênica e proteica desse simportador. As espécies de Palaemon apresentaram os maiores limites de tolerância ao aumento da salinidade, assim como exibiram uma maior capacidade hiporegulatória a longo prazo (120 h) comparada aos representantes de Macrobrachium. Dentre as espécies de Macrobrachium, os limites de tolerância foram maiores nas espécies diádromas do que nas hololimnéticas. Os parâmetros LSS50, osmolalidade e [Cl-] da hemolinfa demonstraram-se estruturados na filogenia, sendo as semelhanças compartilhadas justificadas pela estreita proximidade entre as espécies. As análises filogenéticas revelaram que a capacidade hiper-regulatória da [Cl-] da hemolinfa foi correlacionada com a expressão gênica do simportador NKCC nas brânquias, enquanto que a síntese proteica do NKCC parece estar associada à hiper-regulação da osmolalidade da hemolinfa. A avaliação da localização do NKCC por imunofluorescência demonstrou que o simportador está distribuído em ambas as células que compõem o epitélio das brânquias, as células pilares e células do septo intralamelar. A localização na porção inferior das franjas e no corpo da célula pilar e por toda célula do septo não diferiu entre as espécies, e também não difereiu entre as condições controle e a curto e longo prazo em salinidade elevada. Esses resultados em conjunto sugerem a importância do NKCC também na captação de sal pelas brânquias. Houve um aumento da síntese proteica do NKCC nas brânquias dos representantes de Macrobrachium, exceto M. potiuna, quando em salinidade elevada. Observou-se que este aumento é explicado pela proximidade filogenética entre as espécies. Não houve mudança na transcrição de RNAm para o NKCC apesar do aumento na síntese proteica, o que sugere uma possível regulação pós-transcricional. A reconstrução da história evolutiva da osmorregulação, incorporando o conceito de filofisiologia, revelou a existência de mecanismos em nível molecular, celular e sistêmico que evoluíram acompanhando os eventos cladogenéticos dos Palaemonidae durante a irradiação e ocupação de diferentes nichos osmóticos. / Owing to their evolutionary history, the shrimp family Palaemonidae includes species from widely distinct osmotic environments. Whether from marine, estuarine, or fresh waters, inhabiting stable or variable osmotic niches, these shrimps maintain the osmotic-ionic concentration of their hemolymph independently of the concentration of the external medium. These species hyper-regulate hemolymph osmolality and ions in dilute medium and fresh water and hypo-regulate this fluid in concentrated medium or seawater. The gill epithelium constitutes an important interface of ion transport, and its constituent ionocytes express an ensemble of ion transporters that enable active transepithelial ion movements. The Na+, K+, 2Cl- cotransporter (NKCC) is thought to play a significant role in compensatory salt secretion. Species representative of the marine habitat (Palaemon) were collected from estuaries and tidal pools; diadromous species from the fresh water habitat (Macrobrachium) were collected near the mouths of rivers that flow into the sea, while hololimnetic species were collected in continental streams lacking the influence of brackish waters. The critical upper salinity limits (LSS50) of the marine species P. northropi and P. pandaliformis and the diadromous freshwater species M. acanthurus, M. olfersi, M. amazonicum that depend on brackish water for complete larval development, and the hololimnetic M. potiuna and M. brasiliense that complete their reproductive cycle entirely in fresh water were established. Our objectives were to characterize the mechanisms of hyper-regulation (control condition 18 S P. northropi, 17 S P. pandaliformis, fresh water <0.5 S for Macrobrachium) and hypo-regulation [short-term (24 h) and long-term 120 h) at salinities corresponding to 80% of LSS50] of hemolymph osmolality and [Cl-], gene and protein expression, and NKCC localization for immunofluorescence in the gill ionocytes; (ii) the existence of a phylogenetic pattern in these parameters; and (iii) to test hypotheses for a salinity effect on the evolution of the gene and protein expression of this symporter. The species of Palaemon had the highest tolerance limits to increased salinity, and also exhibited a greater hypo-regulatory capacity for long-term acclimation compared to the species of Macrobrachium. Among the Macrobrachium species, the LSS50 were higher in the diadromous species than in the hololimnetic species. The parameters LSS50 and osmolality and [Cl-] of the hemolymph were phylogenetically structured, similarities being shared by closely related species. The hyper-regulatory capacity of hemolymph [Cl-] correlated with NKCC gene expression in the gills, while NKCC protein synthesis appears to be associated with hyper-regulation of hemolymph osmolality. Immunofluorescence analysis showed that the NKCC was located in both cell types that constitute the gill epithelium, the pillar cells and the septal cells. The location of the NKCC in the lower flanges and perikarya of the pillar cells and throughout the septal cells, did not differ among species, and also did not differ among control conditions or short and long-term exposure at high salinity. These results together also suggest the importance of the NKCC in salt uptake by the gills. When in high salinity there was an increase in NKCC protein synthesis in the gills of the Macrobrachium species, except for M. potiuna. This increase can be explained by the phylogenetic proximity among those species, which excludes adaptive inferences. There was no change in NKCC mRNA transcription, which suggests possible post-transcriptional regulation. The reconstruction of the evolutionary history of osmoregulation, incorporating the concept of phylophysiology, revealed the existence of mechanisms at the molecular, cellular and systemic levels that have evolved accompanying the cladogenetic events of the Palaemonidae during their radiation and occupation of different osmotic niches.

As propriedades bioquímicas da (Na+,K+)-ATPase branquial do siri eurialino Callinectes danae aclimatado à salinidade de 15 o/oo foram estudadas. A análise do gradiente de centrifugação em sacarose revelou a presença de um único pico entre 30-35% de sacarose, com uma boa correlação entre as atividades PNFFase a ATPase totais e (Na+,K+)-ATPase. A atividade residual observada na presença de ouabaína 3 mM sugere a presença de outros sistemas de enzimas atuantes. A eletroforese em condições desnaturantes nos microsomas de brânquias de C. danae em animais recém-coletados em salinidade de 33 o/oo (não aclimatados) e de aclimatados a salinidades de 15 e 33 o/oo por um período de 10 dias mostrou a presença de pequenas diferenças nos padrões eletroforéticos das diferentes amostras. A análise por Western blot mostrou um aumento significativo da proporção relativa da subunidade alfa da (Na+,K+)-ATPase em relação à proteína total na fração microsomal do tecido branquial de animais aclimatados à salinidade de 15 o/oo quando comparados aos animais aclimatados a 33 o/oo. Entretanto, proporções similares de subunidade alfa foram observadas para amostras de animais recém-coletados a salinidade de 33 o/oo e aclimatados a 15 o/oo. A estimulação da atividade (Na+,K+)-ATPase pelo ATP ocorreu através de uma curva de saturação monofásica apresentando interações sítio-sítio (nH=1,2), com V= 298,8 ± 16,7 U/mg, com K0,5 de 174,2 ± 9,8 uM. A estimulação da atividade ATPase da (Na+,K+)-ATPase por íons Mg2+ (V= 299,16 ± 14,06 U/mg; K0,5= 767,31 ± 36,06 uM), íons Na+ (V= 309,0 ± 15,8 U/mg; K0,5= 7,8 ± 0,4 mM), íons K+ (V= 300,6 ± 15,3 U/mg; K0,5= 1,63 ± 0,08 mM) e íons NH4+ (V= 345,1 ± 19,0 U/mg; K0,5= 6,0 ± 0,3 mM) ocorreu através de interações sítio-sítio. A atividade da enzima foi modulada sinergisticamente pelos íons K+ com atividade máxima variando de 300,6 ± 15,3 U/mg para 514,6 ± 26,2 U/mg, na ausência e na presença 50 mM de íons NH4+, respectivamente. Além disso, foi observado um significativo aumento na afinidade aparente da enzima pelo íon K+ da ordem de 10 vezes (diminuiu de 1,6 ± 0,08 mM para 0,157 ± 0,008 mM). Similarmente ao observado para os íons K+, o íon NH4+ estimulou sinergisticamente a atividade da enzima na presença de diferentes concentrações de íons K+. A estimulação da atividade da enzima pelo íon NH4+ também ocorreu através de interações cooperativas entre os sítios. Embora tenha sido observado um aumento da atividade específica da enzima de 345,1 ± 19,0 U/mg para 516,8 ± 27,9 U/mg, não foram observadas variações significativas nos valores de nH e K0,5 com o aumento da concentração de íons K+. A ouabaína inibiu cerca de 90% da atividade ATPase total. A inibição pela ouabaína apresentou valor de KI de 45,09 ± 2,51 uM. O ortovanadato também inibiu atividade (Na+,K+)-ATPase na mesma faixa (90%) através de uma curva de inibição monofásica, com valor de KI da ordem de 1,31 ± 0,06 uM. O emprego de bafilomicina A1, tapsigargina e teofilina, juntamente com a ouabaína, na atividade ATPase total descartam a presença de V-ATPase, Ca2+-ATPase ou fosfatase, respectivamente. Apesar da inibição por oligomicina corresponder a menos de 3,7%, esse valor aparentemente sugere a presença de uma F0F1-ATPase. Além disso, a inibição por ácido etacrínico, em conjunto com os experimentos de estimulação por da atividade ATPase da enzima por íons Na+ sugere fortemente a presença de uma K+-ATPase. A (Na+,K+)-ATPase hidrolisou o substrato PNFF obedecendo à cinética Michaeliana com velocidade de V= 102,9 ± 4,3 U/mg e KM= 1,7 ± 0,1 mM. Já a estimulação da atividade K+-fosfatase da enzima por íons Mg2+ (V= 93,7 ± 2,3 U/mg; K0,5= 1,40 ± 0,03 mM), K+ (V= 94,9 ± 3,5 U/mg; K0,5= 2,9 ± 0,1 mM) e NH4+ (V= 106,2 ± 2,2 U/mg; K0,5= 9,8 ± 0,2 mM) seguiu uma cinética cooperativa, sugerindo a presença de múltiplos sítios de ligação. Entretanto, a atividade K+-fosfatase não foi estimulada sinergísticamente na presença de íons K+ mais NH4+. Os íons sódio (KI= 22,7 ± 1,7 mM) e ortovanadato (KI= 28,1 ± 1,4 nM) inibiram completamente a atividade fosfatase total através de uma única curva de inibição. / The biochemical properties of the (Na+,K+)-ATPase from the gill microsomal tissue of the euryhaline, marine, swimming crab Callinectes danae, acclimated to 15 0/00 salinity, were investigated. Sucrose gradient centrifugation analyses revealed a unique peak, between 30-35% sucrose, coincident with the total PNPPase, ATPase, and (Na+,K+)-ATPase activities. The residual activity observed in the presence of 3 mM ouabain suggests the existence of other enzyme systems. Electrophoresis under denaturing conditions, using material from fresh-caught crabs (33 o/oo salinity, not acclimated), and from crabs acclimated to 15 or 33 o/oo salinity, for 10 days, revealed differences in migration pattern. Western blot analyses showed a significant increase in the amount of (Na+,K+)-ATPase alpha-subunit relative to total protein, for crabs acclimated to 15 o/oo compared to those acclimated to 33 o/oo salinity. However, the proportion of alpha-subunit in samples from fresh-caught crabs acclimated to 33 o/oo and those acclimated to 15 o/oo salinity was similar. (Na+,K+)-ATPase activity was stimulated by ATP and showed a single saturation curve, exhibiting site-site interactions (nH=1.2), with V= 298.8 ± 16.7 U/mg, and K0.5= 174.2 ± 9.8 uM. Stimulation of the ATPase activity by Mg2+ (V= 299.16 ± 14.06 U/mg; K0.5= 767.31 ± 36.06 uM), Na+ (V= 309.0 ± 15.8 U/mg; K0.5= 7.8 ± 0.4 mM), K+ (V= 300.6 ± 15.3 U/mg; K0.5= 1.63 ± 0.08 mM) and NH4+ ions (V= 345.1 ± 19.0 U/mg; K0.5= 6.0 ± 0.3 mM) occurred through site-site interactions. (Na+,K+)-ATPase activity was synergistically modulated by K+ ions, maximum activity varying from 300.6 ± 15.3 U/mg to 514.6 ± 26.2 U/mg, in the absence and presence of 50 mM NH4+ ions, respectively. K+ ions induced a 10-fold increase in enzyme apparent affinity (from 1.6 ± 0.08 mM to 0.157 ± 0.008 mM). As for K+ ions, NH4+ synergistically stimulated enzyme activity in the presence of variable K+ concentrations. The stimulation by NH4+ ions exhibited cooperative, site-site interactions. Although an increase in specific activity from 345.1 ± 19.0 U/mg to 516.8 ± 27.9 U/mg was seen, no significant changes in nH and K0.5 were observed. Ouabain inhibited total ATPase activity by about 90%, showing a KI= 45.09 ± 2.51 uM. Orthovanadate also inhibited the (Na+,K+)-ATPase with a KI of 1.31 ± 0.06 uM. Although the inhibitory effect of oligomycin was minimal (3.7%), this inhibition may suggest F0F1-ATPase activity. The inhibition by ethacrynic acid, in association with Na+ ion stimulation of the ATPase activity, suggests the presence of a K+-ATPase. The (Na+,K+)-ATPase hydrolyzed PNPP (K+-phosphatase activity) obeying Michaelian kinetics, with V= 102.9 ± 4.3 U/mg and KM= 1.7 ± 0.1 mM. The stimulation of K+-phosphatase activity by Mg2+ (V= 93.7 ± 2.3 U/mg; K0.5= 1.4 ± 0.03 mM), K+ (V= 94.9 ± 3.5 U/mg; K0.5= 2.9 ± 0.1 mM), and NH4+ ions (V= 106.2 ± 2.2 U/mg; K0.5= 9.8 ± 0.2 mM) following cooperative kinetics, suggests multiple binding sites. K+-phosphatase activity, however, was not synergistically stimulated by K+ and NH4+. Sodium ions (KI= 22.7 ± 1.7 mM), and orthovanadate (KI= 28.1 ± 1.4 nM) totally inhibited the total phosphatase activity.

Climate change predictions suggest that the continent most vulnerable to climate change is Africa. The impacts of potential changes which include increases in air temperatures and rainfall variability are negative with potential species extinctions projected throughout southern Africa. A number of climate models have been applied to examine the consequences of climate change for ranges of South African animal species. One such model frequently predicted range shifts from west to east, which is realistic considering the marked aridity gradient in an east-west direction across the country, but the authors suggested that these shifts may not be as marked if species are able to use physiological and behavioural methods to adapt to an increase in aridity. Information on the degree to which behavioural and physiological flexibility affect species range in southern Africa is scant which is surprising given its importance with regard to climate change. Thallomys nigricauda occurs along an east-west aridity gradient in southern Africa, inhabiting mesic, semi-xeric and xeric regions. One would expect phenotypic flexibility in physiological and behavioural traits in response to the diverse environmental conditions to be related to the success and range of the species. The wide distribution and arboreal habits, suggesting that T. nigricauda is exposed to greater extremes of temperature than fossorial rodents, makes T. nigricauda an ideal species to test this assumption. Hence I expected that T. nigricauda would exhibit variation in physiological and behavioural traits measured along an aridity gradient. This has important implications in predicting the survival of small mammal species in the light of climate change in southern Africa. Thallomys nigricauda were live-trapped in winter 2006 and 2007 and summer 2007 using Elliot traps in three sites: mesic site Weenen Game Reserve (KwaZuluiv Natal Province, South Africa); semi-xeric site Haina Game Farm (Botswana) on the northern boundary of the Central Kalahari Desert and xeric site Molopo Nature Reserve (southern Kalahari savannah, North-West Province, South Africa). I studied the home-range size of T. nigricauda by radiotracking 12 males and 16 females in winter 2006, 2007 and summer 2007. Home ranges were estimated using 100% and 95% minimum convex polygons and 95% and 50% fixed kernels. Home ranges varied widely, from 166 to 80199m2 for males and from 46 to 8810m2 for females. Males had larger home ranges than females, which supports a promiscuous mating system reported for the species. Although range size was reduced in both sexes in winter, this was not significant. I found no significant difference in home range size along the aridity gradient. It is suggested that a combination of precipitation, habitat productivity and breeding system influences the size of home range of the species, and that this species displays phenotypic flexibility in terms of its behavioural responses to these factors. I measured the urine concentrating ability (UCA), as indicated by urine osmolality and relative medullary thickness (RMT), and water turnover rate (WTR) of T. nigricauda. There was no significant difference in RMT between sites or sex and no difference in osmolalities when site, season and sex were taken into account. In addition, specific WTR was not significantly influenced by season. Lack of significant differences could be the result of the high degree of individual variation in the traits measured, an indication of the flexibility in UCA and WTR. However, higher urine osmolality and lower WTR’s were recorded in the dry winter months. I quantified the thermal environment perceived by a small, arboreal, mammalian endotherm using a number of methods at three study sites in winter and summer. Our area of interest was how well these methods accurately portrayed the actual temperatures that small mammals are exposed to. Temperature differences between the methods were largest during the midday, when temperatures were highest. All methods recorded a greater range of temperatures during photophase than during scotophase. Black-bulb and model temperatures produced more accurate, rapid measurements when compared to measurements produced by direct temperature recording devices, particularly during photophase, when solar radiation is the major influence of heating. Other methods lagged behind black-bulb measurements. Although the mean temperatures of some of the methods were significantly different, there was a high degree of correlation between all methods, even after randomization and generation of 25% and 10% subsamples. Computed thermal indices and blackbulb temperatures produced similar thermal profiles. In studies requiring accurate time series measurements, it is suggested that black-bulb or copper models be employed rather than direct temperature recording devices. Simpler measurement devices would suffice for studies requiring an estimate of the temperature variation and trends in the microclimate of small mammalian endotherms, particularly arboreal or cavity dwelling species. In the wild, across an aridity gradient, I measured abdominal body temperarture (Tb) of T. nigricauda using implanted iButtons®. All but three T. nigricauda displayed significant 24 h Tb rhythmicity. The Tb range for free-living T. nigricauda was 32.33-40.63 oC (n = 13) and 32.69-40.15 oC (n = 17) in winter and summer respectively. Although there was variation in Tb profiles, T. nigricauda generally displayed a bimodal distribution of Tb, with high and low Tb values during scotophase and photopase respectively. Body temperature range was significantly greater in winter, when T. nigricauda reduced its minimum Tb. It was shown that the maximum amplitude of circadian rhythms of body temperature was on average 259.6% of expected values. To determine the extent to which the microclimate of T. nigricauda cavities assists in the maintenance of Tb, I measured the temperatures of cavities across the gradient, providing an indication of the degree of buffering provided by refugia. I measured the temperatures of shallow and deep regions of cavities using iButtons® in summer and winter and recorded operative and shade temperatures for comparison. Compared with operative temperature, cavities had stable microclimates, displaying smaller ranges in temperature. Mean minimum and maximum cavity temperatures differed significantly to operative temperature and between seasons, whereas there was no significant difference between shallow and deep measurements in cavities. Differences in the buffering capacities of the cavities between seasons were not significant. To determine whether T. nigricauda alter its length of exposure in response to lower ambient temperatures in winter as a means of maintaining Tb, I measured the activity of T. nigricauda, defined as the proportion of fixes outside the home cavity of the individual. Males spent a greater proportion of the active phase away from their home cavity in summer, and significantly in winter when compared with females, but there were no differences between seasons. It is suggested that T. nigricauda realize energy savings by lowering its Tb during their rest phase during the day, allowing them to maintain nocturnal activity and overall energy balance. Thus, besides the larger male home range, a result of the reproductive pattern, the physiological and behavioural traits of T. nigricauda measured in this study did not differ between aridity sites or seasons. The results of this study, in highlighting the variation in physiological and behavioural responses of subpopulations of T. nigricauda to diverse conditions, suggest that this variation is due to phenotypic flexibility. Understanding the extent and nature of this flexibility is critical to our comprehension of the consequences climate change. By defining the presence and extent of intraspecific variation in physiology and behaviour, this study resolved the necessary first step towards this understanding for the widely distributed T. nigricauda in southern Africa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Made available in DSpace on 2016-06-02T19:29:14Z (GMT). No. of bitstreams: 1 2200.pdf: 10791536 bytes, checksum: b6e0bb5ddedaa9d93d62362f66b60224 (MD5) Previous issue date: 2008-08-29 / Financiadora de Estudos e Projetos / The relationship between functional morphology of the gills, osmoregulatory physiology, and habitats of the freshwater stingrays (family Potamotrygonidae) was investigated. Potamotrygonid gills have a rather unique external and internal anatomy and organization compared with other marine and/or euryhaline rays. The filaments on the hemibranchs are usually longer and numerous in the second arch. A protuberance was observed on the leading edge of the filaments. The epithelium that covers the gill filaments and lamellae is composed primarily of pavement cells (PVCs), mucous cells (MCs) and chloride cells (CCs). The PVCs showed PAS-positive reactivity. In addition, studies using transmission electron microscopy (TEM) indicate that PVCs possess subapical secretory granules or vesicles that contain mucous material. Large mucous cells were observed with Alcian blue and PAS reaction suggesting the presence of acid and neutral mucopolysaccharides, respectively. Particular attention was focused on the chloride cells. Na+/K+ -ATPase-rich cells (chloride cell, CC-NKA) were frequently found on the trailing edge and in the interlamellar spaces. They were also found on the lamellae, although generally towards the base. The number of CC-NKA and Na+/K+ -ATPase activity were greatest in arch IV compared with the other branchial arches. The basolateral membrane of the chloride cell does has moderate infoldings, and they are likely the site of Na+/K+ -ATPase activity. A surprising result was observed in Potamotrygon sp., in which chloride cells were arranged in large groups in the interlamellar region, not observed in other potamotrygonid species. This multicellular complex of chloride cell is certainly unusual, and may provide a micro-environment suitable to ion uptake from the acidic and ion-poor water of the Rio Negro basin. Potamotrygonid stingrays exhibit typical teleostean body fluid chemistry. These results were analyzed based on the organism-environment interaction. Amazonian rivers, such as Rio Amazonas, Rio Negro, and Rio Tapajós are spatially heterogeneous in their physical and chemical features. In this regard, it is apparent that some distribution patterns of the family Potamotrygonidae may be related to the type of water (e.g white, black and clearwater). The hydrographic barrier hypothesis was tested in Potamotrygon sp. In this ray, plasma [Na+], [Cl-], osmolality and kidney Na+/K+ -ATPase activity decreased after acclimatization to water of the Rio Branco compared to Rio Negro-acclimatized animals. These findings suggest that whitewater-associated changes on the ion and plasma osmolality are due to reduction in the renal Na+/K+ -ATPase activity resulting in an ion loss to the environment. In our biogeographic scenario, some water types may act as an expressive hydrographic barrier for the isolation of endemic potamotrygonid species. On the other hand, Paratrygon aiereba, a widespread stingray that lives in white, clear and blackwaters in the Amazon basin exhibited some physiological differences related to the aquatic environment. Plasma osmolality, urea and ion concentration were higher in whitewater, as compared to blackwater rays. This fact may be explained as an example of phenotypic plasticity, usually expressed in aquatic animals in environments with different aquatic compositions. / Foram analisadas as relações entre a morfologia funcional das brânquias, fisiologia osmorregulatória e habitats das arraias de água doce da família Potamotrygonidae. A organização geral das brânquias dos potamotrigonídeos é semelhante aos demais elasmobrânquios. As hemibrânquias dos potamotrigonídeos possuem entre 74 a 103 filamentos. Em cada filamento observa-se uma protuberância, cujo epitélio é constituído pelas mesmas células diferenciadas que revestem os filamentos e as lamelas dos demais elasmobrânquios: células pavimentosas, células mucosas e células cloreto. As células pavimentosas (CPVs) são PAS-positivas evidenciando a síntese de mucosubstâncias neutras. Estudos em microscopia eletrônica de transmissão (MET) evidenciam a presença de pequenas vesículas subapicais contendo material electrondenso nas CPVs. As células mucosas são grandes e possuem reação Alcian blue e PAS-positivas sugerindo produção de mucosubstâncias ácidas e neutras, respectivamente. As células cloreto imuno-positivas para a Na+/K+-ATPase (CC-NKA) são mais freqüentemente nos espaços interlamelares principalmente no 4º arco branquial. A intensa imunomarcação na periferia citoplasmática das CCs-NKA e os estudos em MET demonstram a presença de moderadas invaginações na região basolateral das células cloreto. Em Potamotrygon sp. (&#8776;arraia cururu, espécie nova) as CCs-NKA agrupam-se em complexos multicelulares, os quais podem ser importantes sítios para absorção de íons a partir de um ambiente extremamente ácido e pobre em sais, como as águas do Rio Negro. As concentrações dos íons e uréia nos compartimentos corporais suportam a semelhança entre o sangue dos potamotrigonídeos e dos teleósteos de água doce. Estes resultados foram analisados com base na natureza da interação organismo-ambiente dos potamotrigonídeos, pois muitos dos rios amazônicos (Amazonas, Negro e Tapajós) são espacialmente heterogêneos do ponto de vista físico-quimico devido às suas origens geológicas. Sugere-se que os padrões de distribuição das arraias de água doce podem estar associados ao tipo água (branca, preta e clara). Com base nas diferenças físicas e químicas entre os rios, a hipótese da barreira hidrográfica foi testada em Potamotrygon sp., uma espécie endêmica do Rio Negro. Esta arraia quando exposta às águas do Rio Branco apresenta redução significativa nas [Na+], [Cl-], osmolalidade e atividade da Na+/K+-ATPase renal. A redução na NKA renal pode ter provocado a perda desnecessária de íons, e consequentemente uma falha nos processos osmorregulatórios. No contexto biogeográfico, explorar um ambiente que impõe limites fisiológicos poderá se tornar uma barreira geográfica para a distribuição dessa espécie. Por outro lado, exemplares de Paratrygon aiereba coletados no Rio Negro e Rio Solimões/Amazonas sugerem a presença de estratégias diferenciais (plasticidade fenotípica) para adaptação em diferentes habitats, os quais incluem ajustes osmorregulatórios de acordo com as características físicas e químicas da água.

Made available in DSpace on 2016-06-02T19:29:17Z (GMT). No. of bitstreams: 1 2471.pdf: 5609066 bytes, checksum: 5eb83354923bf2ef6bb644d15b85bbd2 (MD5) Previous issue date: 2009-06-05 / Universidade Federal de Sao Carlos / Aluminium is very toxic to fish. Biomarkers have been using to indicate water contamination, however, there are few studies concerning tropical fish species. So, the neotropical fish Prochilodus lineatus, were exposed to aproximately 200 &#956;g.L-1 of dissolved Al em acid pH (Al group), only to acid water (pH group) or to water with pH 7.0 (CTR group) for 6, 24, 96h and 10 days to evaluate its effects on haematological, osmo-ionic, metabolic and endocrine parameters besides morphological analysis by light and electronic microscopy. Fishes exposed to Al have all haematological and metabolic parameters increased when compared to CTR group. Osmo-ionic parameters were significantly smaller in fishes exposed to Al as much as the Na+/K+-ATPase enzyme and the CC number smaller Light microscopy reveal that fish exposed to Al in all experimental periods showed lesions widely distributed in the tissue and considered more severe when compared to fish from other groups. In the Al group, the main alterations found were hyperplasia of the epithelial cells, lamellar fusion and hypertrophy of mucous cells. In the animals of pH group, the main alteration was hipertrophy of epithelial cells, especially of the CC. Scanning electron microscopy allowed to see great disorganization in the lamellae of the fishes exposed to Al, detachment of branchial epithelium and reduction of the interlamellar space. Chloride cells with sponge aspect were noted in the pH and Al groups, besides alteration in the microcript of the pv in both groups. Transmission electron microscopy showed that animals from Al group have more CC displaced to the tip of the lamellae and that these CC have a less dense cytoplasm, with a smaller quantity of tubules and mitochondrias and nuclei with altered shapes. The thickness of the lamellae is bigger because of the swollen pv and the interlamellar spaces are reduced. There are, also, more vacuoles in the cytoplasm of these CC. Results allowed to conclude that exposure to Al in acid medium impairs mostly the hyperegulation capacity of the fish, since it affects the active uptake of ions in the gills and also caused serious structural alterations in the organ, leading to functional problems, jeopardizing the survival of the fishes in such situation. / O alumínio é muito tóxico para os peixes. Biomarcadores tem sido usados para indicar essa contaminação, entretanto são poucos os trabalhos com espécies tropicais. Assim, juvenis do peixe neotropical Prochilodus lineatus foram expostos a aproximadamente 200 &#956;g.L-1 de Al dissolvido em pH ácido (grupo Al), somente pH ácido (grupo pH) ou à água com pH 7,0 (grupo CTR) por 6, 24, 96h e 10 dias para avaliar seus efeitos em parâmetros hematológicos, osmo-iônicos, metabólicos e endócrino, além dos parâmetros morfológicos, por meio de MO, MEV, MET, AFCC e EDS das brânquias. Os peixes expostos ao Al tiveram os parâmetros hematológicos e metabólicos aumentados em relação ao grupo CTR. Os parâmetros osmo-iônicos foram significativamente menores nesses peixes assim como a enzima Na+/K+-ATPase e o número de CC nos mesmos animais. A MO revelou que os peixes expostos ao Al mostraram lesões mais amplamente distribuídas nas brânquias e consideradas mais severas, quando comparados aos demais grupos, sendo que as principais alterações foram hiperplasia do epitélio branquial, fusão lamelar e hipertrofia das células mucosas. Nos animais expostos ao pH a principal alteração foi a hipertrofia celular, principalmente das CC. A MEV permitiu destacar o extenso desarranjo nas lamelas dos peixes dos grupos Al, descamação do epitélio além da redução do espaço interlamelar e fusão lamelar. CC com aspecto esponjoso foram notadas nos grupos pH e Al além da alteração no padrão das microcriptas das célula pv em ambos os grupos. Animais expostos ao Al mostraram ainda AFCC menores em relação aos CTR e o EDS indicou picos de Al maiores nesses grupos. A MET mostrou que os animais expostos ao Al têm mais CC deslocadas para as pontas das lamelas e que essas células apresentam o citoplasma menos denso, com uma menor quantidade de túbulos e mitocôndrias e núcleos formas alteradas, além de mais vacúolos. A espessura das lamelas é maior devido ao inchaço das pv, reduzindo o espaço interlamelar. Os resultados permitiram concluir que exposições ao Al em meio ácido prejudicam principalmente a capacidade de hiperregulação dos peixes afetando a tomada ativa de íons nas brânquias além de sérias alterações estruturais nas brânquias, comprometendo a sobrevivência dos animais nessas situações.