Avian Evolutionary Genomics: Studies of Ficedula Flycatchers

In this thesis, different genetic tools are used to investigate both natural variation and speciation in the Ficedula flycatcher system: pied (Ficedula hypoleuca) and collared (F. albicollis) flycatchers. The molecular evolution of a gene involved in postnatal body growth, GH, has shown high degree of conservation at the mature protein between birds and mammals, whereas the variation observed in its signal peptide seems to be adaptive in pied flycatcher (I & II). Speciation is the process by which reproductive barriers to gene flow evolve between populations, and understanding the mechanisms involved in pre- and post-zygotic isolation have been investigated in Ficedula flycatchers. The Z chromosome have been suggested to be the hotspot for genes involved in speciation, thus sequencing of 13 Z-linked coding genes from the two species in allopatry and sympatry have been conducted (III). Surprisingly, the majority of Z-linked genes seemed to be highly conserved, suggesting instead a potential involvement of regulatory regions. Previous studies have shown that genes involved in hybrid fitness, female preferences and male plumage colouration are sex-linked. Hence, three pigmentation genes have been investigated: MC1R, AGRP, and TYRP1. Of these three genes, TYRP1 was identified as a strong candidate to be associated with black-brown plumage variation in sympatric populations, and hence is a strong candidate for a gene contributing to pre-zygotic isolation (IV). In sympatric areas, where pied and collared flycatchers have overlapping breeding areas, hybridization sometimes occurs leading to the production of unfit hybrids. By using a proteomic approach a novel expression pattern in hybrids was revealed compared to the parental species (V) and differentially expressed proteins subsequently identified by sequence similarity (VI). In conclusion, the Z chromosome appears to play an important role in flycatcher speciation, but probably not at the coding level. In addition the novel expression patterns might give new insights into the maladaptive hybrids.

Genetic regulatory networks in avian B cells

Biology is turning into an information science. The science of systems biology seeks to understand the genetic networks that govern organism development and functions. In this study the chicken was used as a model organism in the study of B cell regulatory factors. These studies open new avenues for plasma cell research by connecting the down regulation of the B cell gene expression program directly to the initiation of plasma cell differentiation. The unique advantages of the DT40 avian B cell model system, specifically its high homologous recombination rate, were utilized to study gene regulation in Pax5 knock out cell lines and to gain new insights into the B cell to plasma cell transitions that underlie the secretion of antibodies as part of the adaptive immune response. The Pax5 transcription factor is central to the commitment, development and maintenance of the B cell phenotype. Mice lacking the Pax5 gene have an arrest in development at the pro-B lymphocyte stage while DT40 cells have been derived from cells at a more mature stage of development. The DT40 Pax5-/- cells exhibited gene expression similarities with primary chicken plasma cells. The expression of the plasma cell transcription factors Blimp-1 and XBP-1 were significantly upregulated while the expression of the germinal centre factor BCL6 was diminished in Pax5-/- cells, and this alteration was normalized by Pax5 re-introduction. The Pax5-deficient cells further manifested substantially elevated secretion of IgM into the supernatant, another characteristic of plasma cells. These results for the first time indicated that the downregulation of the Pax5 gene in B cells promotes plasma cell differentiation. Cross-species meta-analysis of chicken and mouse Pax5 gene knockout studies uncovers genes and pathways whose regulatory relationship to Pax5 has remained unchanged for over 300 million years. Restriction of the hematopoietic stem cell fate to produce T, B and NK cell lineages is dependent on the Ikaros and its molecular partners, the closely related Helios and Aiolos. Ikaros family members are zinc finger proteins which act as transcriptional repressors while helping to activate lymphoid genes. Helios in mice is expressed from the hematopoietic stem cell level onwards, although later in development its expression seems to predominate in the T cell lineage. This study establishes the emergence and sequence of the chicken Ikaros family members. Helios expression in the bursa of Fabricius, germinal centres and B cell lines suggested a role for Helios in the avian B-cell lineage, too. Phylogenetic studies of the Ikaros family connect the expansion of the Ikaros family, and thus possibly the emergence of the adaptive immune system, with the second round of genome duplications originally proposed by Ohno. Paralogs that have arisen as a result of genome-wide duplications are sometimes termed ohnologs – Ikaros family proteins appear to fit that definition. This study highlighted the opportunities afforded by the genome sequencing efforts and somatic cell reverse genetics approaches using the DT40 cell line. The DT40 cell line and the avian model system promise to remain a fruitful model for mechanistic insight in the post-genomic era as well.

Kanainfluenssa

Summary: Avian influenza

Lintuinfluenssa : kirjallisuuskatsaus ja villilintujen alustava kartoitustutkimus Suomessa 2003

Summary: Avian influenza : literature review and pilot survey in wild birds in Finland in 2003

Eksoottisten lintujen kulkuteillä

Kirjallisuusarvostelu

Siiveniskuja vuosimiljoonien takaa

Kirjallisuusarvostelu

Isolation and Characterization of Ovomucin - a Bioactive Agent of Egg White

The hen’s egg is a source of new life. Therefore, it contains many biologically active compounds. In addition to being a very nutritious food and also commonly used in the food industry due to its many techno-functional properties, the egg can serve as a source of compounds used as nutra-, pharmaand cosmeceuticals. One such interesting compound is ovomucin, an egg white protein responsible for the gel-like properties of thick egg white. Previous studies have indicated that ovomucin and ovomucin-derived peptides have several different bioactive properties. The objectives of the present study were to develop isolation methods for ovomucin, to characterize the structure of ovomucin, to compare various egg fractions as sources of ovomucin, to study the effects of various dissolving methods for ovomucin, and to investigate the bioactive properties of ovomucin and ovomucin-derived peptides. A simple and rapid method for crude ovomucin separation was developed. By using this method crude ovomucin was isolated within hours, compared to the 1-2 days (including a dialysis step) needed when using several other methods. Structural characterization revealed that ovomucin is composed of two subunits, α- and β-ovomucin, as egg white protein formerly called α1-ovomucin seemed to be ovostatin. However, it might be possible that ovostatin is associated within β- and α-ovomucin. This interaction could even have some effect on the physical nature of various egg white layers. Although filtration by-product fraction was a very prominent source of both crude and β-ovomucin, process development has reduced its amount so significantly that it has no practical meaning anymore. Thus, the commercial liquid egg white is probably the best option, especially if it generally contains amounts of β-ovomucin as high as were found in these studies. Crude ovomucin was dissolved both by using physical and enzymic methods. Although sonication was the most effective physical method for ovomucin solubilisation, colloid milling seemed to be a very promising alternative. A milk-like, smooth and opaque crude ovomucin suspension was attained by using a colloid mill. The dissolved ovomucin fractions were further tested for bioactive properties, and it was found that three dissolving methods tested produced moderate antiviral activity against Newcastle disease virus, namely colloid milling, enzymatic hydrolysis and a combination of sonicaton and enzymatic hydrolysis. Moreover, trypsin-digested crude ovomucin was found to have moderate antiviral activity against avian influenza virus: both subtype H5 and H7.

Effects of algal turbidity on foraging and antipredator behaviour of the three-spined stickleback (Gasterosteus aculeatus)

The aim of this thesis was to examine how aquatic organisms, such as fish, behave in an altered environmental condition. Many species of fish use vision as their primary tool to gain information about their surrounding environment. The visual conditions of aquatic habitats are often altered as a result of anthropogenic disturbance, such as eutrophication that initiates algal turbidity. In general, turbidity reduces the visibility and can be hypothesized to have an influence on the behaviour of fish. I used the three-spined stickleback (Gasterosteus aculeatus) as a model species and conducted four studies in the laboratory to test how algal turbidity affects its behaviour. In this thesis, two major behavioural aspects are discussed. The first is antipredator behaviour. In study I, the combined effects of turbidity and shoot density on habitat choice (shelter vs open) behaviour was tested on a group of sticklebacks (20 fish) in the presence and absence of piscivorous perch (Perca fluviatilis). In study II, I examined the behavioural responses of feeding sticklebacks when they were exposed to the sudden appearance of an avian predator (the silhouette of a common tern, Sterna hirundo). The study was done in turbid and clear water using three different groups sizes (1, 3 and 6 fish). The second aspect is foraging behaviour. Study III & IV focused on the effects of algal turbidity on the foraging performance of sticklebacks. In study III, I conducted two separate experiments to examine the effects of turbidity on prey consumption and prey choice of sticklebacks. In this experiment turbidity levels and the proportion of large and small prey (Daphnia spp.) were manipulated. In study IV, I studied whether a group of six sticklebacks can distribute themselves according to food input at two feeding stations in a way that provided each fish with the same amount of food in clear and turbid water. I also observed whether the fish can follow changes in resource distribution between the foraging patches. My results indicate an overall influence of algal turbidity on the antipredator and foraging behaviour of sticklebacks. In the presence of a potential predator, the use of the sheltered habitat was more pronounced at higher turbidity. Besides this, sticklebacks reduced their activity levels with predator presence at higher turbidity and shoot density levels, suggesting a possible antipredator adaptation to avoid a predator. When exposed to a sudden appearance of an avian predator, sticklebacks showed a weaker antipredator response in turbid water, which suggests that turbidity degrades the risk assessment capabilities of sticklebacks. I found an effect of group size but not turbidity in the proportion of sticklebacks that fled to the shelter area, which indicates that sticklebacks are able to communicate among group members at the experimental turbidity levels. I found an overall negative effect of turbidity on food intake. Both turbidity and changes in the proportion of prey sizes played a significant role in a stickleback’s prey selection. At lower turbidity levels (clear <1 and 5 NTU) sticklebacks showed preferences for large prey, whereas in more turbid conditions and when the proportion of large to small prey increased sticklebacks became increasingly random in their prey selection. Finally, my results showed that groups of sticklebacks disperse themselves between feeding stations according to the reward ratios following the predictions of the ideal free distribution theory. However, they took a significantly longer time to reach the equilibrium distribution in turbid water than in clear water. In addition, they showed a slower response to changes in resource distribution in a turbid environment. These findings suggest that turbidity interferes with the information transfer among group foragers. It is important to understand that aquatic animals are often exposed to a degraded environment. The findings of this thesis suggest that algal turbidity negatively affects their behavioural performance. The results also shed light on the underlying behavioural strategies of sticklebacks in turbid conditions that might help them adapt to an altered environmental situation and increase their survival. In conclusion, I hold that although algal turbidity has detrimental effects on the antipredator and foraging behaviour of sticklebacks, their behavioural adjustment might help them adapt to a changing environment.