Doctoral Theses / Doktorsavhandlingar Zoologiska institutionen

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Age-class interactions in Atlantic salmon and brown trout: Effects on habitat use and performance
(2010-05-06T12:42:48Z) Kaspersson, Rasmus
This thesis investigates the underlying mechanisms and the density-regulatory effects of age-class interactions, using juvenile Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.) as study species. Field experiments were performed in streams along the western coast of Sweden, in which densities of older age-classes were reduced and the response on young-of-the-year habitat use and performance (growth, movement and survival) was observed (Papers I and II). Observational data from 159 trout populations was extracted from the Swedish Electro-fishing Register to test the generality of age-class competition (Paper III) and observations in controlled artificial stream environments were used to establish the underlying mechanisms with regard to habitat use and behavioural interactions (Papers IV and V). The combined findings of these studies show that age-classes of stream-living salmonids compete for limited resources in the stream habitat. This competition favours old individuals, although the behavioural observations of Paper V suggest that their competitive benefit may decrease at increasing densities of young-of-the-year fish. Density-reductions of older cohorts in field increased the growth of young-of-the-year trout, an effect that was observed at the later part of the growth season (Papers I and II). The observational data-set (Paper III), provided further evidence of the prevalence of inter-cohort competition, reflected as a negative association between density of older cohorts and young-of-the-year body-size, in the same magnitude as on an intra-cohort level. In accordance with previous studies, juvenile salmon and trout were segregated in the stream habitat, with young-of-the-year individuals using shallow, low-velocity, habitats close to the spawning area while older cohorts were positioned in deep, high-velocity, areas (Papers II and IV). However, when experimentally reducing the density of older cohorts in field and lab (Papers II and IV), this spatial pattern was shown to be an effect of habitat exclusion rather than size-dependent habitat preference, as suggested in previous studies, with subsequent negative effects on young-of-the-year foraging activity (Paper IV). Thus, this finding provides a potential underlying mechanism to the negative effect on young-of-the-year performance presented in Papers I, II and III. From an applied point of view, the findings of this thesis highlight the importance of taking age-class interactions into account when investigating density-dependence and habitat use among stream-living salmonids. The findings also suggest that marginal stream habitats may be essential during the first months after emergence by acting as refuges from inter-cohort competition, thus emphasizing the importance of maintaining and restoring these habitats in the wild.
Alkali-containing aerosol particles-release during biomass combustion and ambient air concentrations
(2009-01-22T09:31:18Z) Kovacevik, Borka
Air pollution in the form of particles has considerable influence on human health and climate. Atmospheric aerosol particles arise from direct emissions of particles and from the conversion of certain gases to particles in the atmosphere. They are produced by a number of different sources and have a typical lifetime of about a week in the atmosphere. The chemical composition of the atmospheric aerosol is highly variable in time and space, and the different effects of the aerosol can usually not be understood without taking its multi-component nature into account. The overall aim of this thesis is to contribute to an improved understanding of climate effects and air quality issues related to aerosol particles. The thesis focuses on aerosol particles containing alkali-compounds, and particles produced by biomass combustion are of particular interest. Aerosol mass spectrometer techniques for highly sensitive and selective detection of alkali-containing particles have been further developed and applied in laboratory experiments, in fluidised bed combustion, and in ambient air measurements. The experimental techniques provide the chemical composition of individual aerosol particles with high time resolution and they are well suited for combustion aerosol applications, and for detection of sea salt particles that contain a large fraction of sodium compounds. Laboratory experiments were performed to study the emission of potassium and sodium-containing compounds during rapid pyrolysis of birchwood. The alkali emission during the pyrolysis phase and from ash and char formed at high temperatures was characterized. Studies were also carried out during biomass combustion in a 12 MW (thermal) circulating fluidized-bed boiler. The effect of the addition of chlorine and/or sulphur to the fuel on fly ash composition, deposit formation, and superheater corrosion was investigated. Addition of sulphur and chlorine increased the formation of submicron particles and lead to enhanced deposition of potassium sulphate and chloride. The results compared well with results from earlier laboratory-scale experiments concerning the effects of chlorine and sulphur on potassium chemistry. The findings are of importance for actions aimed at minimizing alkali related corrosion and deposition problems during large-scale biomass conversion. The seasonal variation of the elemental composition of particulate matter in Skopje was studied by chemical analysis of samples collected with impactor technique. Major aerosol components were identified including mineral dust, oil combustion, traffic-related aerosol and secondary sulphate, while a separate biomass burning component could not be identified with the employed methods. Aerosol mass spectrometry was used to study the influence of atmospheric transport patterns and meteorology on alkali concentrations in Gothenburg. The observed potassium and sodium concentrations were concluded to be affected both by emissions from the nearby region and by long-range transport. Sodium-rich sea salt particles were favoured by westerly winds and high wind speeds, and were preferentially observed in air masses originating from the Atlantic. Potassium-rich particles originating from biomass burning were favoured by low temperatures and low wind speeds, and they were most abundant in air masses transported from Eastern and Central Europe.
Biodiversity and genetic patterns in marine invertebrates
(2010-09-17) Vodoti, Emma
Abstract: Systematics based on genetic data has both confirmed and contradicted earlier, morphologically defined species and their relatedness. Morphology does not always correspond to genetic lineages, and this will inevitably affect both traditional systematics as well as biodiversity assessments. My thesis aims to investigate genetic biodiversity in some marine invertebrates, dealing with both species and population (intraspecific) relationships. I discuss gene lineage relatedness in correlation to species morphs and geographical species distribution, which has bearing upon the species recognition problem, the barcoding approach and the meaning of phylogeographic patterns. Results show that morphology does not generally reflect gene lineage relationships in the considered species and that cryptic species are common, mainly in the nemertean groups studied (Oerstedia dorsalis; Cerebratulus spp). Despite having a relatively established status as a species, the horse mussel (Modiolus modiolus) is also found to hold cryptic lineages. Further, the difficulties in species delimitation become apparent, as an almost continuous range of genetic divergence prevails between most of the found clades. The Antarctic krill (Euphausia superba) is the only study species lacking cryptisism, at least in the samples and geographical region studied. The intraspecific genealogies were in this case used to estimate population parameters by means of the coalescent. Despite being an abundant species of modest size, results suggest a low effective population size and a population with common ancestry, most likely capable of migrating over relatively great distances. This stresses that factors such as demography and genetics can provide a preliminary, general base for management issues. I conclude that cryptic species are common, and therefore taxonomic work cannot be isolated from the barcoding quest of summoning genetic data for species identification. Since genetic relationships and morphological traits do not always go hand in hand, a barcoding approach could in some cases be misleading. Further, it is probably impossible to find a universal way of defining, identifying and delimiting species. This thesis illustrates some practical examples of these problems, and suggests that a case-to-case evaluation is likely needed in future taxonomic and phylogenetic efforts.
Chronic stress and intestinal barrier function: Implications for infection and inflammation in intensive salmon aquaculture
(2009-10-09T13:52:59Z) Sundh, Henrik
Aquaculture is a fast growing food-producing sector worldwide. This has increased the awareness among the public as well as governments of the need to secure the welfare and health of farmed fish. Atlantic salmon is, in non-Asian countries, one of the most successful aquacultured species. Despite continuous improvement of husbandry practices and development of new vaccines, both bacterial and viral diseases are main threats to the health of farmed Atlantic salmon. Chronic stress, created by sub- optimal husbandry conditions is believed to be an important factor behind disease outbreaks. The intestinal epithelium of Atlantic salmon is an important organ. Not just in nutrient absorption, but also as a barrier, preventing antigens and pathogens within the intestinal lumen to gain entrance to the host and thereby start an infection. Chronic stress is known to have a negative impact on the intestinal barrier in mammals, a situation that can cause a leaky epithelium and increased bacterial translocation. Stressful husbandry conditions could have a similar effect on the intestine of Atlantic salmon and would thus be a major threat to the welfare of farmed fish. This thesis aimed at elucidating the importance of the intestinal physical and immunological barrier function in disease susceptibility of farmed Atlantic salmon. It further aimed to reveal the impact of key husbandry conditions in Atlantic salmon aquaculture hypothesized to be potential threats to health and welfare of the fish. All husbandry conditions examined, i.e. hyperoxygenation combined with low water flow, low levels of dissolved oxygen and high temperature as well as high fish density in combination with poor water quality was concluded stressful to the fish. These common husbandry practises all generated primary and secondary stress responses such as increased plasma cortisol levels, increased cortisol release rate into the water and a decreased function of all intestinal barriers. Decreased physical barrier was evident in the form of increased paracellular permeability. This was accompanied by increased translocation rates of the pathogen bacteria Aeromonas salmonicida, suggesting an increased risk for bacterial infections in fish under chronic stress. Dysfunction of the intestinal immunological barrier was manifested as impaired expression of the pro-inflammatory cytokine IFN-γ. This down regulation could be the result of an immuno suppressive effect of cortisol but could also be an effect of the decreased physical intestinal barrier resulting in increased leakage of luminal antigens. The decreased expression of IFN-γ could be a sign of decreased cellular immunity which would render an intestinal barrier with reduced ability to cope with virus infections like the IPNV. Chronic stress also resulted in increased homing of neutrophils and signs of sever local inflammation. These findings suggest that stressed fish may experience increased disease susceptibility towards viral pathogens like IPNV as well as an increased risk of developing chronic intestinal inflammation. Intestinal barrier function was further evaluated as a new scientific tool for assessment of potential threats to welfare in aquaculture. The intestine was demonstrated to be a sensitive indicator of stress at times when no or only minor differences in primary stress responses was observed In conclusion, common husbandry may constitute a threat to health and welfare of farmed fish and intestinal barrier function can serve as a valuable tool for assessing potential stressful husbandry conditions in the future.
Control of bioluminescence. Operating the light switch in photophores from marine animals
(2009-02-02T07:42:11Z) Krönström, Jenny
Physiologically controlled photocytes, capable of producing bioluminescence, are a common feature in the ocean among animals ranging from cnidarians to fish. The aim of this thesis was to study and compare the nature of this control, in both distantly and closely related species from the groups Teleostei Crustacea, and Cnidaria. This was done using histochemistry and electron microscopy to reveal the internal morphology of the different photophores and to identify the location of nerves and signalling substances inside these organs. Moreover, luminescence responses of isolated photocytes, photophores or live animals, exposed to drugs with effects on adrenergic, noradrenergic, 5-hydroxytryptaminergic and nitric oxide signalling mechanisms, were measured. Nitric oxide donors had modulating, primarily quenching, effects on the luminescence from the fish species Argyropelecus hemigymnus and Porichthys notatus and the krill Meganyctiphanes norvegica. However, a few of the A. hemigymnus photophores, and a part of the P. notatus response were potentiated when using nitric oxide donors. The variety in nitric oxide responses was reflected by the presence of nitric oxide synthase-like material in different cell types, including neurons, photocytes and lens/filter cells, in the photophores from the studied fish species and Meganyctiphanes norvegica. Capillary sphincter cells and capillary endothelia contained nitric oxide synthase-like material in Meganyctiophanes norvegica photophores. Moreover, varicose nerve fibres, containing 5-hydroxytryptamine, followed the capillaries and reached the sphincter cells, suggesting that nitric oxide and 5-hydroxytryptamine may interact and control the resistance for haemolymph flow in the photophores, but other mechanisms are also discussed in the thesis. Contractile properties of the sphincter structures, and possibly the endothelial cells, were supported by the presence of muscle-like filaments in the sphincter structures and filamentous actin in both sphincter and endothelial cells. Relaxation of sphincters and capillaries may increase the flow of oxygenated haemolymph to the light-producing cells, thus stimulating or facilitating luminescence. Further indications for this scenario were a stimulation of luminescence by muscle relaxing substances and a quenching of the 5-hydroxytryptamine stimulated luminescence by a muscle contracting substance. Attempts to study an adrenergic mechanism in cnidarians failed for unknown reasons. In conclusion, it was shown that a nitric oxide signalling system is present in the photophores from several luminescent species. The variety of nitric oxide responses, as well as the variety of morphological arrangements and patterns of innervation in the studied photophores emphasise the biodiversity of bioluminescence.
Ecological genetics of inbreeding, outbreeding and immunocompetence in Ranid frogs
(2008-11-21T09:29:09Z) Sagvik, Jörgen
Using artificial fertilization, I crossed frogs from different populations to evaluate fitness consequences for the offspring from an inbreeding-outbreeding perspective, and to evaluate quantitative genetic effects on immunocompetence against a fungal pathogen (Saprolegnia). Crosses between closely situated populations of different sizes generated contrasting results for the effects of outbreeding on offspring traits between populations and life history stages, emphasizing the importance of epistatic effects and the difficulties of relying on generalizations when making conservation decisions (e.g., regarding translocations). Experimental infection of frog eggs from six populations with Saprolegnia fungus showed a significant family effect on the degree of infection of eggs and embryos, in particular at lower fertilization success and with a significant temperature × population interaction effect. A paternal genetic effect on fungus resistance was found using a half-sib split design. Furthermore, relatively more eggs were infected when fertilized by sperm from the same, in contrast with a different population. However, there was no evidence for a stronger effect in isolated island populations. Although the mechanistic underpinnings remain unknown, these results suggest substantial levels of genetic variation in resistance to Saprolegnia in natural populations within and among populations. We also found that pre-hatching exposure to Saprolegnia dramatically reduced the size at metamorphosis in the absence of further exposure to the fungus, possible as a delayed effect of impaired embryonic development. However, in contrast to some other amphibians, induced hatching in response to Saprolegnia could not be confirmed. In conclusion, the results suggest that frog populations are genetically diverse even at small geographic scale with frequently strong and unpredictable consequences of in- and outbreeding for the response to stressors.
Egg size evolution and paternal care in pipefishes.
(2010-08-30) Braga Gonçalves, Inês
In this thesis I explore how sexual selection, sexual conflicts, coevolution with parental care and an environmental selective agent (hypoxia) affect the evolution of different egg characteristics and embryo survival and size in pipefishes (Syngnathidae). In the broad-nosed pipefish, Syngnathus typhle Linnaeus, both sexes prefer to mate with large partners; large females produce more and larger eggs and large males can care for more young, and, as shown in this thesis, invest more per embryo and provide better oxygenation during brooding. However, if given time to assess their partners prior to mating, females deposit significantly more proteins into eggs when they mate with non-preferred (smaller) males. This result suggests that females are able to improve offspring fitness by compensating for small males’ lower paternal care quality, thus reducing effects of differences in care between small and large males. The spherical shape common to fish eggs minimizes the ratio between surface area and volume. As embryo respiration is assumed to be proportional to egg volume and oxygen availability to be restricted by the surface of the egg, this shape may be unfavourable for larger eggs. In S. typhle, both small eggs and hypoxia (low ambient oxygen levels) during brooding resulted in smaller embryos, but the negative effect of hypoxia on embryo size was similar for small and large eggs. In brooding males, within-pouch oxygen levels were found to be consistently lower than those of the surrounding environment, both in high and low ambient oxygen conditions indicating a limited paternal ability to maintain high levels of oxygenation to the embryos. When comparing pipefish species with and without brood pouches, the species with pouches were found to suffer, on average, greater embryo mortalities during brooding. However, under hypoxia embryo survival and size were negatively affected both in species with and without brood pouches. Between species, large egg size resulted in significantly larger offspring, but had a negative effect on embryo survival, compared to species that produce small eggs. This thesis contributes to the understanding of egg size evolution in relation to hypoxia and paternal care in syngnathids. The studies presented here suggest that the evolution of brood pouches carries costs in terms of embryo oxygenation. Furthermore, the evolution of more complex types of brooding has potentially enabled phenotypic variation to arise in the quality of male care, in turn selecting for flexible female reproductive investment with stronger selection on egg size evolution in species with brood pouches, and greater female control of egg quality.
Environmental influences on the behavioural ecology of juvenile salmonids – the importance of rearing density
(2010-01-10T13:41:41Z) Brockmark, Sofia
Background and aims: Early environmental conditions are known to influence the phenotypic development of animals, including behavioural alterations. The overall aim of this thesis is to investigate the effects of density, social stability and structural complexity on growth, behaviour, and survival in the wild, using juvenile brown trout (Salmo trutta) and Atlantic salmon (Salmo salar) as model species. The result can add to the basic knowledge in this research area and can also be used to enhance the welfare and fitness of released hatchery-reared fish. Methods: In papers II-IV sea-ranched trout and salmon were reared from early life stage in conventional hatchery tanks with modified physical structure and/or density. Hatchery fish were measured for growth, studied for individual and social behaviours, and analysed for fin-erosions and smolt-status. In paper I wild-caught juvenile trout were used for behavioural studies. In papers II-IV fish from different treatment groups were also released in natural streams to investigate treatment effects on survival and growth in the wild. Results and conclusions: My results collectively show clear positive effects of reduced rearing density, whereas the effects of structure were unclear and harder to interpret. In papers II-IV reduction of conventional hatchery densities generally increased growth rate in the hatchery and post-release survival. Salmonids reared at reduced densities were more dominant in competition for food, consumed more novel prey, escaped faster to refuges after a predator attack and located more food in a maze, compared to fish from higher densities. At time of parr-smolt transformation, salmon reared at reduced densities had less fin erosions and were more fully smolted than salmon reared at higher densities. Taken together, the results presented in this thesis clearly show that reduced rearing densities facilitate the development of adaptive individual and social behaviour in salmonids, resulting in increased growth and survival after release in nature. In paper I wild trout in familiar groups were more vigilant, responded faster to a simulated predator attack and consumed more food compared to trout in unfamiliar groups. These novel results suggest that social stability confer immediate fitness benefits, i.e. higher probability of survival under conditions of high predation risk. In summary, my results show that conventional rearing methods in supplementary hatcheries do not prepare fish adequately for life in the wild and could be improved considerably, with density reduction as one key factor. Incorporating behavioural aspects in supplementary rearing methods is also important from an ethical point of view. However, there is a limit to what can be accomplished with improving supplementary hatchery rearing methods. Hatchery rearing should therefore be viewed as a complement rather than an alternative to habitat restoration.
Environmental, nutritional and endocrine regulation of metabolic processes in fish
(2011-11-18) Kullgren, Andreas
Due to seasonal variations in temperature and food availability, fish in temperate regions should be able to make metabolic adjustments to ensure that enough energy is available for the maintenance of basal processes. The major aim of this thesis was to elucidate how the physiology and lipid metabolism of salmonid fish is affected by temperature and food availability, and to clarify aspects of the endocrine control of lipid metabolism. In this thesis, the effects of increased temperature or reduced food availability were studied in salmonids by employing a non-prejudiced metabolomics approach to assess the physiological responses. Detailed information on the abundance of specific amino acids, lipid classes, fatty acids and other metabolites in tissue extracts and plasma was obtained by nuclear magnetic resonance (NMR) based metabolomics. NMR-based metabolomics were successfully employed and proved to be applicable to studying metabolic fluxes in fish, providing data on novel and integrated responses. The results show similar changes in lipid metabolism during food deprivation and elevated temperature. The observed responses included increased plasma very-low-density lipoprotein (VLDL) and unsaturated fatty acids (FAs) concurrent with decreased high-density lipoprotein (HDL) and choline. The changes during starvation also involved changes in amino acids and glycogen that indicate that amino acids are used for gluconeogenesis in the liver to preserve glycogen stores. Growth hormone (GH) has both lipolytic and lipogenic effects. To further elucidate the mechanisms of GH action on salmonid lipid metabolism, the effects of GH in vivo on the transcription of several key lipid metabolism enzymes in various tissues were investigated. GH inhibited the hepatic expression of lipoprotein lipase (LPL) thereby decreasing hepatic lipid uptake. Hormone-sensitive lipase (HSL) mRNA expression was not increased by GH in any of the studied tissues, suggesting that the well-known GHinduced lipolysis is regulated on posttranslational levels in rainbow trout. The regulation lipid metabolism in salmonids was further investigated by studying direct effects of FAs and ghrelin on freshly isolated cells from mesenteric adipose tissue and liver. FAs elicited acute negative effects on lipid storage by decreasing lipid uptake via LPL activity in adipose cells as well as by stimulating lipolysis of stored triglycerides (TG) in liver cells. Together the results presented in this thesis shows that elevated, suboptimal temperature and nutritional may have propound effects on important processes as growth, food intake and the metabolome of salmonid fish, and may lead to a negative energy balance. Metabolic changes may be mediated by hormonal and nutrient factors acting at gene expression or enzyme activity level. The results may contribute to better understand lipid deposition patterns in farmed fish and potential effects of climate change on salmonids in the wild and in aquaculture.
Evolution of annelid diversity at whale-falls and other marine ephemeral habitats
(2009-09-03T11:21:24Z) Wiklund, Helena
When whales die and sink to the sea-floor, they provide a sudden, enormous food supply to organisms in the vicinity. At first, larger mobile scavengers remove the flesh, but also when only the bones remain, the whale-fall can still sustain macrofauna communities for several years. Some organisms have adapted so well to this kind of food source that they would have problems living elsewhere, e.g. species within the polychaete genus Osedax which have developed a root system which can bore into the bones to reach nutrients. Other organisms are not so specialized on the bones, but rather on the sulphur-oxidizing filamentous bacterial mats covering the bones. Polychaetes from several families have been observed grazing the bacterial mats, and some of the species reported from whale-falls have also been found in other ephemeral, chemosynthetic habitats like e.g. hydrothermal vents, cold seeps and beneath fish farms, where filamentous mat-forming bacteria also occur. In this thesis, nine new species from two polychaete families are described from whale-falls and fish farms in Scandinavian waters, and from whale-falls and sunken wood off the Californian coast. Their phylogeny is investigated using molecular data. The genetic data are further used to separate morphologically cryptic species from ephemeral habitats in different ocean basins.
From Proteomic Analysis to Biomarker Application - Studies of Carbonyl Reductase in Fish
(2011-09-22) Albertsson, Eva
Many anthropogenic substances are present in the aquatic environment, but there is limited information on how this combination of chemicals affects exposed wildlife. To assess the impact of chemicals, a biomarker approach is frequently applied in environmental biomonitoring programmes. In this thesis, proteomic analysis was used as a hypothesis generator for biomarker development in fish. Two-dimensional gel electrophoresis was performed on a liver fraction from rainbow trout (Oncorhynchus mykiss) that were caged downstream of a sewage treatment plant, and it was found that a protein identified as carbonyl reductase was induced. This was confirmed with qPCR, indicating that carbonyl reductase mRNA expression levels were markedly induced by the same effluent and also by a second investigated sewage treatment plant effluent of a similar character. More advanced sewage treatment technologies reduced carbonyl reductase mRNA expression, indicating that fish that were exposed to effluents of conventionally treated sewage have induced hepatic carbonyl reductase expression. To obtain more information regarding the possible inducers of carbonyl reductase, several different single-substance exposure studies were performed with rainbow trout in controlled aquaria systems. The results demonstrated that hepatic carbonyl reductase expression was not induced by the steroids that were tested but by an aryl hydrocarbon agonist and a pro-oxidant. Others have observed substances with the same mode of action in the effluents of Swedish sewage treatment plants. To promote the use of carbonyl reductase as a biomarker, a method for measuring enzymatic activity was developed in the sentinel coastal fish species eelpout (Zoarces viviparus), which is included in the Swedish national integrated fish-monitoring program. The protocol for carbonyl reductase enzymatic activity combined with qPCR analyses in eelpout were applied on a comprehensive field material, including sites with different grades of pollution among the Swedish, Danish and German coasts. Carbonyl reductase activity and mRNA did not show any site-specific differences between the polluted and reference sites. Therefore, it can be concluded that the induction of carbonyl reductase appears to be a promising biomarker for assessing exposure and the possible impact of sewage treatment plant effluents in caged fish. However, more research is required before and if carbonyl reductase can be used in biomonitoring programmes as a biomarker to assess the impact of toxic chemicals in wild fish populations.
Growth hormone and somatolactin function during sexual maturation of female Atlantic salmon
(2008-10-31T09:10:52Z) Benedet, Susana
Background and aims: The growth hormone-insulin-like growth factor I (GH-IGF-I) system is known to act during sexual maturation of female salmonids, but the specific roles are not known. Somatolactin (SL) is a pituitary hormone closely related to GH and is only found in fish. In some species, including salmonids, there are two forms, SLa and SLß. The SL receptor (SLR) has recently been cloned and phylogenetic analysis shows that it is similar to previously cloned GH receptors (GHRs) of non-salmonids. The ligand-specificity of the GHR/SLR is unclear. Little is known about the role of the SLs in sexual maturation of fish. The aim of this thesis has been to increase our knowledge about the regulatory role(s) of both the GH-IGF-I system and of SLs during sexual maturation in female Atlantic salmon. Methods: The cDNA sequences of Atlantic salmon GHRs (two isoforms), SLR, as well as SLa and SLß were obtained with the goals of carrying out a phylogenetic analysis, and of developing molecular tools for analysis of mRNA levels using real time quantitative PCR (RTqPCR). The roles of GH, IGF-I and SL were examined in a 17-month long study on one sea winter Atlantic salmon females. mRNA expression levels of ovarian components of the GH-IGF-I system and SLR and pituitary GH, SLa and SLß were studied by RTqPCR. Levels of GH and IGF-I in plasma, and of GH in the pituitary were measured by radio-immunoassay. Results and Conclusions: The phylogenetic analysis (Paper I and II) of the cloned sequences reveals the placement of Atlantic salmon GHR in the GHR type II clade and SLR in the controversial GHR type I clade (putative SLRs). Concurrent analyses of pituitary GH mRNA levels, GH protein and plasma GH in the same individual fish demonstrates the complex dynamics of the GH system, which is inhibited by a continuous light. Papers III and IV confirm that there is an active GH-IGF-I-gonad axis in the female Atlantic salmon that appears to be functional at the start of exogenous vitellogenesis, final oocyte growth, spawning and possibly during postovulatory events. Evidence has been found for a photoperiod-driven GH-system activation which is initiated in January with stimulation of GH secretion from pituitary somatotropes. The role of this activation of the GH system in late winter/early spring appears to be the reversal of a prior plasma IGF-I and ovarian IGF-I mRNA downregulation driven by an unknown factor(s). This downregulation in IGF-I is thought to inhibit somatic cell proliferation. The activation of the GH-IGF-I-gonadal system also appears to limit energy allocation to gonadal growth. This series of events involving the GH-IGF-I system appears to take place during the so-called spring window of opportunity and it is the first time this has been described. The GH-IGF-I system also appears to have an important role during final oocyte growth, spawning and post-spawning events. SLa and SLß are both actively regulated during sexual maturation and could have several roles, such as signaling the status of visceral fat reserves during the spring window of opportunity, signaling lipid metabolic status before the onset of anorexia, involvement in Ca mobilization during vitellogenesis and/or control of lipid metabolism in lieu of GH during the final stages of oocyte growth.
Haematopoietic and proteomic responses to wounding stress in the common sea star Asterias rubens
(2008-10-01T06:53:44Z) Holm, Kristina
Dissertation Abstract Holm, Kristina (2008). Haematopoietic and proteomic responses to wounding stress in the common sea star Asterias rubens. Department of Zoology, University of Gothenburg, Sweden Many species of echinoderms have a remarkable ability to regenerate lost tissues, including the sea star A. rubens. The initial step in regeneration is wound healing, necessary to prevent disruption of body fluid balance, and to limit the invasion of pathogens. Injury initiates an immune response, where the circulating cells are activated. In general, invertebrates have a well developed innate immune system that is mediated by circulating blood cells. In the sea star these kinds of cells, the coelomocytes, respond with a rapid and massive accumulation at the wound site. The aim of this thesis was to identify and localize the haematopoietic tissues, the source of stem cells for renewal of the coelomocytes, as well as to increase knowledge of response to wounding, with focus on the coelomocytes and their protein expression, in the common sea star A. rubens. Synergistic effects of hypoxia were also investigated. It could be concluded that cells in the coelomic epithelium respond with proliferation when triggered with mitogenic factors and show a protein expression pattern very similar to the pattern of circulating coelomocytes. The shape and behaviour of cells migrating out of the coelomic epithelium show high similarities with the behaviour of coelomocytes, in terms of phagocytosis and network formation. Tiedemann body and axial organ are also proposed as haematopoietic tissues, since a significant increase in proliferation was seen also in these tissues after triggering with mitogens as well as a pattern of protein expression similar to coelomocytes. Total coelomocyte count (TCC), as well as the expression of the heat shock protein (HSP) 70, is known to increase with severe stress in sea stars. The stresses investigated here, wounding together with hypoxia, induced a significant increase in TCC even after 1 hour while 6 hours after wounding TCC had increased approximately two-fold. Western blot analysis revealed highly elevated coelomocyte cytoplasmic HSP70-expression 3 hours after wounding. Non-wounded sea stars exposed to hypoxia and wounded animals kept in normoxia, showed enhanced HSP70 expression only after 24 hours. This synergistic stress response of wounding together with hypoxia may suggest ecological consequences, since the hypoxic areas in the ocean are growing. Protein fractions separated by size from the coelomic fluid of wounded A. rubens, had slightly different effects on coelomocytes/haemocytes from three groups of invertebrates, the mollusc Mytilus edulis, the tunicate Ciona intestinalis and the echinoderm, A. rubens itself, where also effects on explants of coelomic epithelium were examined. The fraction containing proteins of the size 15-70 kDa showed a significant cytotoxic effect on the sea star coelomocytes and tissue samples, but seemed not to be cytotoxic for mussel or tunicate cells. This fraction, with smaller proteins, may contain cytokine-like molecules such as interleukin (IL)-1 and IL-6, but also the invertebrate form of lysozyme, molecules previously described in the sea star. The two-dimensional gel electrophoresis method was developed for analyzing the protein content of coelomocytes. The methodology was optimized in terms of sample preparation, pI interval, gel gradient and staining procedure. The analysis of protein spots using MALDI-TOF/TOF mass spectrometry resulted in 9 identified protein homologues out of 18, of which 6 were found significantly up- or down-regulated. Sample preparation and methodological choices can and should be developed depending on the purpose of a study. The protocol developed here will be useful in future proteomic studies, maybe also for other marine organisms. The databases searched included the sea urchin Strongylocentrotus purpuratus, but the search result show no closer relationship between S. purpuratus and A. rubens, than to other invertebrates or even vertebrates within the database. Future proteomic studies of A. rubens may give valuable information about the wound healing and regeneration processes in sea stars as well as in other animals including humans.
Individual variation in behaviour: personality and performance of brown trout in the wild
(2010-05-07T11:15:43Z) Adriaenssens, Bart
Individuals from the same population often show very different behaviour. These differences, when consistent across time, are referred to as animal personality or behavioural syndromes. Explaining the occurrence of animal personality from an evolutionary perspective has however proven a difficult issue to tackle. This thesis studies aspects of individual behavioural variation and personality in brown trout (Salmo trutta). More specifically, I investigate (1) to what extent variation in behaviour is consistent within and across contexts, (2) environmental and genetic effects on behaviour, (3) how this affects performance in the wild, and (4) whether this understanding can be used to improve rearing methods of supplementary hatcheries. I found brown trout to express a wide variation of behaviours and provide evidence that much of this behavioural variation is associated in bigger behavioural syndromes. As a result, separate behaviours of brown trout cannot be considered as isolated units, but combine into clusters that sometimes are associated with non-behavioural measures such as body size or growth rate. Variation was further influenced by both inherited and environmental effects. First, individuals from different maternal and paternal origin differed in size, aggressiveness and response to novel prey or novel food. These results suggest that maternal and/or genetic effects influence behaviour and growth in brown trout (I). Second, reduced rearing densities in a hatchery increased the response to novel prey, food search ability in a maze and predator response (II). And third, hatchery trout were more successful foragers than wild conspecifics, yet showed less repeatable explorative behaviour across time (III). Personality traits were generally poor predictors of growth and survival upon release, suggesting that several behavioural strategies can be successful in nature. Nevertheless, in paper IV, slow exploring individuals grew faster than more bold trout. Furthermore, parr reared at reduced densities were twice as likely to survive in the stream as trout reared at high densities. In conclusion, my results contradict simple associations between risk taking behaviour and growth-mortality tradeoffs under natural conditions. This challenges the recent view that individual differences in growth strategies can explain variation in behaviour and suggests more heterogeneous links between personality and life-history in nature (V). In addition, I show that reduced rearing hatchery densities facilitate the development of adaptive behaviour in brown trout, a finding that may have implications for current rearing methods in supplementary hatcheries.
Infidelity in Birds – Causes and Consequences of Extra-pair Paternity
(2012-04-13) Augustin, Jakob
Forty years ago, more than 90% of bird species were classified as monogamous and not very exciting systems for studies of e.g. sexual selection. Since then, the discovery of extra-pair paternity (EPP) in more than 75% of surveyed monogamous bird species has made avian monogamy, and the interaction between social and genetic mating systems in general, a challenging and attractive area of research. Despite three decades of research on EPP in birds, however, many questions and controversies remain unresolved. This thesis contributes to the understanding of mechanisms and adaptive reasons, primarily from the female’s perspective, for the highly diverse frequencies of EPP in birds. First, in a population of the common redshank (Tringa totanus), a wader for which the genetic mating system has not been described previously, a surprising absence of EPP is demonstrated (I). Presumably, some female pre- or postcopulatory resistance to extra-pair fertilisations is present. The potential mechanisms and adaptive significance of this is discussed in relation to redshank ecology and behaviour. In the three following papers (II-IV), assumptions and predictions of hypothesised female benefits from EPP are addressed. In sand martins (Riparia riparia), there were no indications that extra-pair fertilisations resulted in genetic benefits (e.g. heterozygosity or ‘good genes’) (II). Paper III tests an assumption related to the genetic compatibility hypothesis, i.e. that overall heterozygosity leads to increased chick survival; this did not seem to be the case in Kentish plovers (Charadrius alexandrinus). In northern lapwings (Vanellus vanellus), the indirect benefits hypothesis is partly supported by a positive association between EPP and brood sex ratio (IV). As predicted by the differential sex allocation hypothesis, broods with extra-pair offspring contained a higher proportion of sons than broods without extrapair offspring. As for the yet unknown mechanism of sex determination in birds, an unusual case of a fertile, triploid Kentish plover female is presented and discussed with regard to the two present major hypotheses for sexdetermination (VI). Finally, as an alternative or additional interpretation of what appears to be brood sex ratio adjustment by the female, the often neglected effect of differential mortality is discussed (V).
The intestinal epithelium of salmonids : transepithelial transport, barrier function and bacterial interactions
(2006) Jutfelt, Fredrik
The salmonid intestinal epithelium is important for growth and health of the fish. The epithelium is exposed to a multitude of internal and external factors that can influence its function. During the parr-smolt transformation and subsequent seawater transfer, the epithelium adapts for an osmoregulatory role and the fish starts drinking seawater (SW). Endocrine signals increases the intestinal water uptake partly through an up-regulation of Na+,K+-ATPase activity. It is shown that the epithelial paracellular permeability decrease concurrent with the increase in water transport, suggesting that water flow is directed from a paracellular to a more transcellular route. The rational for this could be the increase in epithelial exposure to the environment at SW entrance. Tightening the paracellular route could be a mechanism to reduce paracellular transfer of harmful substances and pathogens. A major salmonid pathogen is the bacterium Aeromonas salmonicida, which cause losses in both aquaculture and in wild populations. It is not known, however, by which route the A. salmonicida enters the fish. A. salmonicida has been positively demonstrated in the intestinal lumen but it has been controversial whether or not the bacteria cross the epithelial barriers. It is demonstrated that A. salmonicida can translocate across the intestinal barrier, indicating the intestine as a functional route for bacterial infection in salmonids. It is concluded that A. salmonicida employs many virulence mechanisms, such as exotoxins, endotoxin and cell bound factors, to disrupts epithelial morphology and function and promote translocation. During the later phases of parr-smolt transformation the epithelial barrier integrity decreased and translocation of pathogens increased. The increased disease susceptibility during this life stage could thus partly be caused by a decreased barrier function. Vegetable lipids are used as replacement for fish oil in salmonid aquaculture, but there are concerns about how the new diets affect the intestinal epithelium. The epithelial functions presently investigated indicate a slight increase in permeability, supporting earlier histological reports of epithelial disruptions but not to the same extent. Nutrient uptake and barrier function during the parr-smolt transformation was significantly improved by a vegetable lipid-containing diet, indicating that this inclusion may be beneficial in the freshwater (FW) stage. The fatty acid profile of the natural diet for salmonids in FW is more similar to a blend of vegetable oils than to the profile of marine feed ingredients, routinely used in salmonid aquaculture. This may be the rationale for the positive effects. Salmon fed sunflower oil, however, showed long term elevation of plasma cortisol levels indicating a chronic stress. As chronic stress is known to depress immune function, specific vegetable lipids potentially stressful to the fish may also affect their health and welfare. Thus, while vegetable lipids at certain life stages are feasible substitutes for fish oil, possible long term stress effects by vegetable oils should be considered. In conclusion, the salmonid intestinal epithelium is a sensitive and dynamic tissue which is affected by external factors, such as pathogen bacteria, environment and diet, but which also can be endogenously regulated to compensate for this disturbance.
Melanosome transfer, photoreception and toxicity assays in melanophores
(2009-10-02T09:24:19Z) Hedberg, Daniel
Many animals such as fish and frogs have developed the ability to change colour of their skin to adapt to the environment or to signal to other individuals. This ability is due to specialised skin cells called melanophores. Melanophores contain thousands of melanosomes, small membrane-enclosed organelles containing the black or brown pigment melanin. The melanosomes can aggregate to the cell centre rendering the cells pale or disperse throughout the cell to become dark. The intracellular transport of melanosomes is regulated by neuronal or hormonal external stimuli. Fast colour change is achieved by aggregation/dispersion of melanosomes but long-term colour change can also be achieved by melanosome transfer to surrounding skin cells. An amphibian immortalized melanophore cell line was used from the African claw frog, Xenopus laevis to study transfer of melanosomes to co-cultured fibroblasts. Melanosome transfer was observed and up regulated by the hormone α-MSH . The transfer was quantified using light-, fluorescence and electron microscopy. A new and powerful method for transfer experiments was developed. Fluorescent semiconductor nanocrystals, qdots, were used in combination with flow cytometry. The qdots were taken up by the cultured Xenopus laevis melanophores, localised to the melanosomes and transferred to co-cultured fibroblasts. The method is a step towards enabling large scale analysis of pigment transfer. Xenopus laevis melanophores can be cultivated in 96-well culture plates which allow quantification of aggregation or dispersion in a fast and reproductive way. Glyphosate containing herbicides, i.e. Roundup, are commonly used in the world, but some toxic effects have been found on amphibians in vivo and human and mouse cells in vitro. To learn more about potential effects on intracellular transport and the cytoskeleton in animal Roundup, glyphosate, glyphosateisopropylamine and isopropylamine were tested on the transport of melanosomes to the cell centre by spectrophotmetry and by fluorescence microscopy on microtubules and actin filaments. All tested compounds inhibited the aggregation and affected the morphology of the cytoskeleton. The effect was found to be pH dependent. Amphibian melanophores can be regulated directly by light via a melanopsin receptor. Photoreception was found in cultured early embryos of the zebrafish Danio rerio. Light induced dispersion of the melanophores was contrast to what is found at adults when light causes aggregation of the melanosomes due to signals from the CNS. At least one subclass of melanopsin was detected in the zebrafish retinal pigment epithelial cells.
Morphological antipredator adaptations in water fleas
(2009-10-19T10:31:43Z) Lord, Hans
Some Bosmina and Daphnia species have the ability to develop extreme morphological antipredator defences, such as long antennules, high carapaces and helmets. The relative sizes of these plastic traits may differ substantially between populations, and also between individuals within a population, between sexes and during ontogeny. In this thesis I examine how abiotic factors (trophic levels and temperature) and biotic factors (fish and invertebrate predators) affect the size and shape of these traits. In the first two studies, calculations based on experimental results using physical morphological models sinking in glycerine, were used to estimate body drag and energy consumption in Bosmina. Eubosmina longispina, with a low carapace and short antennules, and Eubosmina coregoni gibbera, with a very high carapace and long antennules, were examined. At 5○C, E. c. gibbera had 32-45 % higher body drag than E. longispina. At 20○C the difference was 20-45 %. A model of swimming predicted that, all else being equal, this difference should result in 18-20 % (at 5○C) or 14-16 % (at 20○C) lower swimming speed for E. c. gibbera than for E. longispina. This indicates substantial hydrodynamic costs of the morphological antipredator defences, particularly in low temperatures. The morphological antipredator defences in E. c. gibbera are larger and more variable in females than in males. Male models had lower body drag than models of asexual and sexual females, suggesting that males can swim 14-28 % faster with the same energy consumption. High speed video documentation of swimming E. c. gibbera showed that males advanced 55-73 % further than females in each swimming stroke. Hydrodynamic body drag may therefore have significant implications for swimming and evolution of sexual dimorphism in water fleas. Males may lack the defensive, but hydrodynamically expensive, high carapace of female E. c. gibbera probably because competition over mates favours low body drag. The morphology and size of heads and trunks in Daphnia cristata individuals were studied in twenty lakes in the same drainage basin. The lakes showed a variation in lake total phosphorus and in densities of invertebrate predators and fish. In the end of the summer D. cristata had varying shape and size of their heads. Our results indicate that D. cristata, in nutrient rich lakes, show adaptive morphological defences against both visually hunting and size limited predators. Female D. cristata head shape, in the examined lakes, varied from small and rounded to large and curved. A significant positive correlation between lake total phosphorus and the allometric head coefficients was found, suggesting that the most extremely shaped heads are found in nutrient-rich lakes.
New Insights into the Evolution of Bryozoa- An Integrative Approach
(2011-02-17) Fuchs, Judith
Bryozoa is a group of aquatic, sessile invertebrates with circumglobal distribution and includes about 6000 recent species. Bryozoans have an indirect life cycle with a larval stage that settles and metamorphoses into the adult. Although a bryozoan individual is barely visible with the naked eye, all bryozoans form colonies, which are often macroscopic in size and display a variety of beautiful shapes and forms. Ever since their first scientific description in the 16th century, bryozoan relationships to other animal groups have been enigmatic. Bryozoan morphology and life history show various differences to other invertebrates, so that their closest relatives could not be identified with certainty. Also, a reliable hypothesis about the evolution of the variety of bryozoan larval and adult body forms is greatly in dispute. In this thesis, questions concerning bryozoan evolution are addressed from diverse angles by exploring different life cycle stages and methodological tools. A new phylogeny of Bryozoa based on molecular data is presented. A similar approach is used to investigate the phylogeny of another animal taxon, Entoprocta, which was long thought to be the sister group of Bryozoa. The results reveal that Bryozoa is a natural group with a single origin (monophyletic clade) and that Bryozoa and Entoprocta are not sister groups. Further, gene expression in the larval stage of the bryozoan Bugula neritina was studied and indicates the importance of molecularly pre-patterned blastemic tissues for adult body plan formation. In addition, a new bryozoan species from the West Coast of Sweden is described and a genetic barcode is provided for the new species, which will help to identify this species in the future. The thesis demonstrates that molecular data combined with high taxon sampling are essential to reveal bryozoan phylogenetic relationships and that gene expression studies of the enigmatic taxon Bryozoa are valuable to get insights into the evolution of their life cycle and to contribute to our general understanding of metazoan body plan evolution.
On the regulation of Postprandial Gastrointestinal Blood Flow in Teleost Fish
(2010-05-20T11:28:46Z) Seth, Henrik
Henrik Seth (2010) On the Regulation of Postprandial Gastrointestinal Blood Flow in Teleost Fish Department of Zoology/Zoophysiology, University of Gothenburg, Box 463, 405 30 Göteborg. The regulation of the cardiovascular changes, in particular the increase in gastrointestinal blood flow that follows after feeding has received little attention in teleost fish. Therefore, the aim of the research that led to this thesis was to discern some of the mechanisms behind the postprandial cardiovascular response. Several methods, described within this thesis, were used in order to study, in vivo, the influence of both mechanical as well as chemical stimuli in triggering the increase in gastrointestinal blood flow that occurs after feeding in fish. Furthermore, additional methods, combining in vivo and in situ pharmacology were used to study the regulatory mechanisms in more detail. The results indicate that both mechanical as well as chemical stimuli are important during the postprandial response. Mechanical stimuli within the stomach evoke an increased adrenergic tone and chemical stimuli induce a subsequent hyperemia that is localized within the gastrointestinal tract. The response to chemical stimuli is also influenced by the composition of the diet. Furthermore, even though the extrinsic innervation (sympathetic and parasympathetic) of the gastrointestinal tract is important in controlling the routine tone of the gastrointestinal vasculature, it is of little importance during the postprandial hyperemia. In contrast, the intrinsic innervation (enteric) within the gastrointestinal tract is of fundamental importance to this hyperemia. In addition, the response is most likely modulated, in response to the diet composition, by endocrine and paracrine factors, such as the gastrointestinal hormone cholecystokinin. In conclusion, the regulation of the gastrointestinal vasculature after feeding is very complex and several mechanisms contribute to the cardiovascular response that will depend on the composition of the diet as well as surrounding environmental factors such as temperature, oxygen levels and stress.

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