Doctoral Theses / Doktorsavhandlingar Zoologiska institutionen

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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.
Oxidative Damage in Fish Used as Biomarkers in Field and Laboratory Studies
(2008) Carney Almroth, Bethanie
Many toxic xenobiotics entering into the aquatic environment exert their effects through redox cycling. Oxidative stress, incorporating both antioxidant defences as well as oxidative damage, is a common effect in organisms exposed to xenobiotics in their environment. The studies included in this dissertation evaluate the effects of different types of environmental pollution on oxidative stress biomarkers in teleost fishes. Effects of aging, alone or in combination with oxidative stress, on protein carbonylation were also addressed. Antioxidant enzyme activities were measured in rainbow trout (Oncorhynchus mykiss) caged in a river polluted by sewage treatment plant (STP) effluent as well as highly contaminated sediment, and in corkwing wrasse (Symphodus melops) collected at heavy metal contaminated and PAH contaminated sites. Antioxidant enzymes showed very few changes in these fish. Glutathione levels were affected by STP effluent exposure in rainbow trout and by PAH exposure in corkwing wrasse. Protein carbonylation was elevated in plasma of the corkwing wrasse captured at the heavy metal site and in plasma of rainbow trout caged near the STP effluent. Lipid peroxidation was elevated in the livers of these rainbow trout. Oxidative damage biomarkers were also measured in eelpout (Zoarces viviparus) captured in a polluted harbor, before, during and after a dredging campaign, as well as following an oil spill. Protein carbonyl levels in livers of eelpout were affected by exposure to pollution in the harbor, though this was measured both as increases and as decreases, indicating a complicated relationship between prooxidant exposure and protein carbonyl accumulation. Lipid peroxidation in eelpout was unaffected by pollutant exposure. Western blot analyses of protein carbonylation in corkwing wrasse exposed to heavy metals and in rainbow trout exposed to paraquat (PQ) suggest that albumin may be the plasma protein most likely to undergo carbonylation in these fish. Brown trout (Salmo trutta) were used in a laboratory study to investigate the effects of aging on oxidative stress parameters, i.e. protein carbonylation, 20S proteosome activity and glutathione levels. All parameters were affected by aging, as was the response to PQ exposure, where 1 year old fish were more sensitive than 0+ fish. To conclude, the results presented here indicate that antioxidant enzymes may not provide a good biomarker of exposure to xenobiotics in the field. However, oxidative damage products, i.e. protein carbonyls and lipid peroxidation, seem to be useful as biomarkers. Care should be taken to consider age of the exposed individuals. Oxidative stress is a complex phenomenon to measure in the field. Antioxidant enzyme activities may be altered after an acute exposure and then return to normal levels but damage products can persist even after the initial stress has stabilized.
Relationships between nomenclature, phylogenetics and systematics
(2008-09-01T11:58:15Z) Bertrand, Yann
Systematists have become increasingly aware of the limits imposed by the current system of nomenclature for accurately representing evolutionary relationships and managing efficiently names associated with clades. In reaction, a new system of nomenclature, the PhyloCode is being developed that fully recognizes the historical nature of taxonomy and the importance of the cladistics revolution. As a consequence, questions emerge about the new historical entities of systematics, questions that can be apprehended through the lens of epistemology, philosophy of language and metaphysics. What is the ontological nature of entities that lack any other essential features besides spatiotemporal properties? How to depart from the fixed realm of immutable and transcendental essence into a worldview wherein all biological entities are characterized by their temporality and materiality? What are the consequences of nomenclatural decisions on other sectors of biology? With the ever growing sequencing capacity and tree reconstructing abilities, our conceptualization of phylogenetic relationships is changing at an unprecedented pace. Then it begs the question, what prevents communication break down when the references of clades’ names are changing almost on a daily basis. These are some of the fundamental issues I am tackling in the present work. Addressing the ontological issue, I argue that species and clades are best perceived as mereological sums of individuals, which means that each biological individual is the unique individual composed of all its less inclusive individuals and nothing more. I propose to separate the meanings of “clade” and “monophyletic group”. I suggest to use “monophyletic” for an epithet referring to a defining property of a set (a natural kind) and “clade” for a noun which corresponds to a historical entity (an individual) resulting from evolutionary process. I present the idea that a phyloname is not attached to a single clade but to a natural kind containing as members the clades that would be selected in counterfactual phylogenies. The defining properties of this natural kind are provided by the phylogenetic definition. Finally I stress that taxonomists are also driven by the will to narrate the same sort of history, when they adjust the reference of names in light of new phylogenetic data, which leads me to submit that taxa can also be perceived as narratives.
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.
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.
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.
Predation and shorebirds: predation management, habitat effects and public opinions
(2009-01-09T09:35:38Z) Isaksson, Daniel
Many shorebird populations are rapidly declining and a high nest predation rate is one of the threats facing these populations. Thus, factors that affect predation and how to manage it in an effective way are receiving increased attention. This thesis deals with nest predation in two ground-nesting shorebirds (waders): the Northern lapwing Vanellus vanellus, and the redshank Tringa totanus nesting in coastal pastures. I study how habitat structures affect nest predation and distribution. I also test the effectiveness of two non-lethal methods for managing predation, and survey public attitudes towards predator control and animal conservation. Predators that hunt by sight often search for prey from elevated perches, such as trees, stone walls and fences. Theory suggests that prey visibility depends strongly on predator perch height and distance. I estimated how prey (a mounted bird) visibility depends on predator perch height, distance and vegetation height in coastal pastures. Visibility increases strongly with observer perch height and proximity. For example, from the lowest perch (0.2 m), visibility of the target bird declines to < 5% beyond 20 m distance, but 40% of it remains visible from the highest perch (8 m), even as far as 120 m. The strong increase of prey visibility with predator search height suggests that the removal of predator perches can improve the survival of endangered prey populations in open habitats. Predators such as the hooded crow Corvus cornix use raised structures for perching and to elude lapwing attacks. I find that crows spent more time at or near raised structures than expected and that wader nests were placed farther away from these structures than expected in two out of three years. Waders thus tend to avoid breeding close to raised structures, which therefore reduces the suitable breeding area and probably also the local wader population size. Habitat management is just one technique for reducing nest predation, and apart from lethal predator control, there are several non-lethal methods. I tested the effect of nest exclosures to protect individual wader nests from predation. Protected nests had a higher hatching success than unprotected nests. Protected redshanks suffered increased predation on incubating adults, which often sit on the nest until a predator is close by. These results emphasize the need for caution in the use of nest exclosures, particularly in redshanks and other species with similar incubation behaviours. Exclosures can, however, be a useful management tool in shorebirds that leave their nests early, when an approaching predator is still far away. I also tested predator avoidance of wader eggs by placing mimic eggs injected with an illness-producing substance in artificial nests. Compared to control areas, the daily survival rate was higher for wader nests during the first three weeks in areas with aversive eggs, but there was no difference for the nesting season as a whole. Egg predation by foxes and other nocturnal mammals may have masked a greater aversion effect in avian predators. I suggest that the dose of the illness-producing substance should be increased and the aversion-learning period prolonged in future tests of this potentially useful technique. Predation management sometimes includes lethal predator control, which can be controversial, and knowledge of public attitudes is essential for successful conservation measures. Using a mail survey sent to a representative sample of the Swedish public (1 751 replies) I found that there is support for protecting threatened animals. Although the support for a general control of animals was low, a majority supported several specific reasons for control, including control of animals that pose a risk to threatened species or to traffic. The support for control varied depending on species, being the lowest for raptors and the highest for mice and rats. A majority did not support the use of more costly non-lethal control in place of lethal methods, but urban residents and animal rights supporters were more positive than the others. I conclude that available perches can have significant effects on prey detection and distribution of wader nests. It is possible to reduce nest predation in some shorebird species using non-lethal techniques such as nest exclosures. When using lethal predator control, I suggest that information about the reasons for control as well as the species involved is highly important, especially as in regard lethal control in urban regions.
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.
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.
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.
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.
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.
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.
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.
Systematics of Grania (Clitellata: Enchytraeidae), an interstitial annelid taxon
(2010-02-12T09:55:47Z) De Wit, Pierre
In between the grains of sand on the ocean floor, there exists a world which few people are aware of. Representatives of almost all animal phyla can be found here. The clitellate family Enchytraeidae is in the marine interstitial environment represented in large part by species of a genus called Grania, which are long slender worms found in marine sands throughout the world. This thesis is a study on the systematics of these worms. The body wall of Grania is searched for phylogenetically informative morphological characters. It is found that the cuticular morphological variation seen in naidids is absent, but the collagen fiber thickness varies between Grania species. Also, the circular and outer, triangular longitudinal musculature is reduced compared to that of closely related taxa while the inner, ribbon-shaped longitudinal muscle fibers are well-developed, possibly an adaptation to interstitial life. The Grania-fauna of the Great Barrier Reef is investigated, with four new species described and Grania trichaeta re-described. The phylogenetic position of Grania within the family Enchytraeidae is elucidated by molecular means, where Lumbricillus arenarius is shown to be a close relative of a monophyletic Grania. Within the genus, a molecular phylogeny is inferred of a sample of 19 species, showing considerable morphological homoplasy, while geographical distribution is concordant with the phylogeny. Thus, we combine morphology with geography, while using the DNA-based tree as a backbone constraint, to estimate a phylogeny of all 71 currently described species within the genus. Finally, the genetic variation within Scandinavian species of Grania is studied with the resulting find of a cryptic species, and the realization that although intraspecific variation generally is low, deviant individuals exist. Within this study, we also infer a phylogeny of the Scandinavian species of Grania, which seems to be a monophyletic group, and discuss their morphological character evolution.
Phylogeny and Signal Diversity in Widowbirds and Bishops (Euplectes spp.)
(2010-02-19T08:56:11Z) Prager, Maria
Although sexual selection for elaborate signals is well documented in numerous species, the extreme diversity in signal design and expression in many taxa is largely unexplained. This thesis explores phylogenetic, mechanistic and ontogenetic explanations for divergence in two classic condition-dependent signal traits in the African widowbirds and bishops (Euplectes spp.); elongated black tails (in widowbirds) and patches of bright yellow or red carotenoid coloration (most prominent in bishops). A molecular phylogeny of 33 Euplectes subspecies (representing all 17 species) was derived using parsimony and Bayesian analyses of mitochondrial and nuclear DNA sequences. A consensus tree, or a sample of the most probable Bayesian trees, was then used in parsimony, likelihood and Bayesian reconstructions of ancestral signal states. Specifically, the discrete presence of a nuptial tail (i.e. prenuptial tail moult), continuous tail length, and discrete as well as continuous reflectance-based measures of carotenoid colour hue were analysed. The proximate basis of interspecific colour variation was investigated using High Performance Liquid Chromatography (HPLC) analyses of feather and plasma pigments in five Euplectes species. Finally, the relative importance of nutritional and metabolic constraints behind differential occurrence of C4-keto-carotenoids, and thus red plumage color, in Euplectes, was tested by diet manipulation in a yellow and a red bishop species. Results show monophyly of the genus Euplectes, but not of ‘widowbirds’ or ‘bishops’. Most notably, the red-collared widowbird E. ardens belongs to a clade of short-tailed bishops and not to the ‘true’ widowbirds. Extant Euplectes furthermore derive from ancestors in which breeding males had short (not prenuptially moulted) tails and yellow colour signals. Nuptial tail elongation and red coloration have since evolved at least twice in distinct lineages, possibly as convergent responses to early established and directional sexual selection for increasingly exaggerated quality advertisements. This provides an interesting contrast to several recent findings of labile ornament evolution in birds and other animals. Three different pigment profiles were identified in Euplectes feathers. Yellow colours primarily depend on dietary yellow carotenoids, while red hues result either from addition of metabolically derived red C4-keto-carotenoids, or from high concentrations of dietary and derived yellow pigments. A possible genetic constraint on colour evolution was also identified, as the southern red bishop E. orix, but not the yellow-crowned bishop E. afer, can manufacture red C4-keto-carotenoids (α-doradexanthin and canthaxanthin) from yellow dietary precursors (lutein and β-carotene). Combined with previous work on adaptive signal functions in Euplectes, the phylogenetic and proximate analyses in this thesis provide an unusually complete picture of avian plumage diversification, and a useful framework for further exploration of both genetics and ecology of avian colour signalling.
Studies of fish responses to the antifoulant medetomidine
(2010-03-31T11:02:12Z) Lennquist, Anna
ABSTRACT Growth of marine organisms, fouling, on man-made constructions submerged in the water is regarded as a major problem. For vessels, fouling increases drag and thereby fuel consumption, wherefore antifouling paints are used. Traditionally, they contain toxic compounds, and several of these have unwanted effects in the environment. Today the search for environmentally acceptable and efficient alternatives is intense. Medetomidine, originally used as a veterinary sedative, inhibits barnacle settling at nanomolar concentrations. It is presently under evaluation for use as an antifouling agent. The studies within this thesis were performed to investigate medetomidine responses in fish. The focus was to identify early effects, occurring from low concentrations. Studies have been performed in the species rainbow trout, Atlantic cod, turbot, Atlantic salmon and three spined stickleback. Exposure time vary from 1 up to 54 days, and a set of parameters have been investigated including biochemical biomarkers, growth and related parameters, behaviour and large scale gene expression. Paleness is the most obvious effect of medetomidine in fish and appears from 0.5 to 50 nM, depending on species. Colour was observed and quantified, and the function of melanophores (pigment cells) after long term exposure to medetomidine was investigated. It is suggested that melanophores are functional after treatment, and thus the colour change may be reversible. Although not lethal per-see, paleness may have consequences for fish predator-prey interactions (camouflage), social signalling and UV protection. Medetomidine also showed to affect the activity of the hepatic enzyme Cytochrome P4501A (CYP1A), measured as EROD activity. A minor increase in activity was observed in vivo in several of the investigated species. In vitro, medetomidine showed instead to be a potent inhibitor of EROD activity with median inhibition values (IC50) in the nanomolar range. An inhibited CYP1A activity may interfere with fish detoxification of toxicants abundant in the aquatic environment. No significant effects were found on growth rate, but the results indicate lowered blood glucose levels and decreased liver size after medetomidine treatment and thus a shift in carbohydrate metabolism. The large scale gene expression study revealed no significant differences among treatments. We found no effects on glutathione or glutathione dependent enzymes in any of the studies. In the behavioural studies, fish were less active and had less appetite in medetomidine treatments compared to control. Medetomidine had no effects on investigated antioxidant enzymes and showed no cytotoxicity. Among the responses studied within this thesis, paleness and inhibition of EROD activity are perhaps the most important. These effects appear early and are clear and consistent among several species.
Taxonomy and phylogeny of polychaetes
(2010-05-03T12:26:18Z) Eklöf, Jenny
Polychaetes are a large group of segmented worms that display an enormous morphological diversity. Molecular data has shown in recent years that groups previously thought to be separate from polychaetes are actually part of the group. The relationships within polychaete groups have been difficult to discern, and molecular data only partly corroborate classifications done on morphological grounds. The main focus of this thesis is on Phyllodocidae, a family of polychaetes, and its phylogenetic relationships. Our results show that none of the phyllodocid subfamilies, as previously delineated by morphology, find support from molecular data. Instead groups previously not recognized receive high support. A number of polychaete families are holopelagic, and most of these have been regarded as closely related to phyllodocids. We have found that one of these holopelagic families, Alciopidae, is well nested within the phyllodocids, with its closest sister being Eumida arctica, making the genus Eumida, as delineated today, paraphyletic. Part of this thesis also deals with cryptic species, which means that two or more species are virtually impossible to separate morphologically, but still represent separately evolving lineages, reproductively isolated from each other. We have found that Arctic and boreal populations of Paranaitis wahlbergi belong to two separate species, and the boreal populations are referred to a new species, P. katoi sp. n. We have also found that sympatric populations of Notophyllum foliosum, found in deep and shallow waters are two separate species, morphologically distinguished only by subtle details in their colouration, and the deep form is described as N. crypticum sp. n. A description is also provided for Axiokebuita, previously not found in European waters. Due to delineation problems with the two described species in the genus it was not possible to refer these new specimens to either of them or to a new species. A phylogenetic analysis of molecular data confirms the position of Axiokebuita among scalibregmatids. Part of this thesis also deals with cryptic species, which means that two or more species are virtually impossible to separate morphologically, but still represent separately evolving lineages, reproductively isolated from each other. We have found that Arctic and boreal populations of Paranaitis wahlbergi belong to two separate species, and the boreal populations are referred to a new species, P. katoi sp. n. We have also found that sympatric populations of Notophyllum foliosum, found in deep and shallow waters are two separate species, morphologically distinguished only by subtle details in their colouration, and the deep form is described as N. crypticum sp. n. A description is also provided for Axiokebuita, previously not found in European waters. Due to delineation problems with the two described species in the genus it was not possible to refer these new specimens to either of them or to a new species. A phylogenetic analysis of molecular data confirms the position of Axiokebuita among scalibregmatids.
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.
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.

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