Environmental effects of thermal and radioactive discharges from nuclear power plants in the boreal brackish-water conditions of the northern Baltic Sea

During recent decades, thermal and radioactive discharges from nuclear power plants into the aquatic environment have become the subject of lively debate as an ecological concern. The target of this thesis was to summarize the large quantity of results obtained in extensive monitoring programmes and studies carried out in recipient sea areas off the Finnish nuclear power plants at Loviisa and Olkiluoto during more than four decades. The Loviisa NPP is located on the coast of the Gulf of Finland and Olkiluoto NPP on that of the Bothnian Sea. The state of the Gulf of Finland is clearly more eutrophic; the nutrient concentrations in the surface water are about 1½ 2 times higher at Loviisa than at Olkiluoto, and the total phosphorus concentrations still increased in both areas (even doubled at Loviisa) between the early 1970s and 2000. Thus, it is a challenge to distinguish the local effects of thermal discharges from the general eutrophication process of the Gulf of Finland. The salinity is generally low in the brackish-water conditions of the northern Baltic Sea, being however about 1 higher at Olkiluoto than at Loviisa (the salinity of surface water varying at the latter from near to 0 in early spring to 4 6 in late autumn). Thus, many marine and fresh-water organisms live in the Loviisa area close to their limit of existence, which makes the biota sensitive to any additional stress. The characteristics of the discharge areas of the two sites differ from each other in many respects: the discharge area at Loviisa is a semi-enclosed bay in the inner archipelago, where the exchange of water is limited, while the discharge area at Olkiluoto is more open, and the exchange of water with the open Bothnian Sea is more effective. The effects of the cooling water discharged from the power plants on the temperatures in the sea were most obvious in winter. The formation of a permanent ice cover in the discharge areas has been delayed in early winter, and the break-up of the ice occurs earlier in spring. The prolonging of the growing season and the disturbance of the overwintering time, in conditions where the biota has adjusted to a distinct rest period in winter, have been the most significant biological effects of the thermal pollution. The soft-bottom macrofauna at Loviisa has deteriorated to the point of almost total extinction at many sampling stations during the past 40 years. A similar decline has been reported for the whole eastern Gulf of Finland. However, the local eutrophication process seems to have contributed into the decline of the zoobenthos in the discharge area at Loviisa. Thermal discharges have increased the production of organic matter, which again has led to more organic bottom deposits. These have in turn increased the tendency of the isolated deeps to a depletion of oxygen, and this has further caused strong remobilization of phosphorus from the bottom sediments. Phytoplankton primary production and primary production capacity doubled in the whole area between the late 1960s and the late 1990s, but started to decrease a little at the beginning of this century. The focus of the production shifted from spring to mid- and late summer. The general rise in the level of primary production was mainly due to the increase in nutrient concentrations over the whole Gulf of Finland, but the thermal discharge contributed to a stronger increase of production in the discharge area compared to that in the intake area. The eutrophication of littoral vegetation in the discharge area has been the most obvious, unambiguous and significant biological effect of the heated water. Myriophyllum spicatum, Potamogeton perfoliatus and Potamogeton pectinatus, and vigorous growths of numerous filamentous algae as their epiphytes have strongly increased in the vicinity of the cooling water outlet, where they have formed dense populations in the littoral zone in late summer. However, the strongest increase of phytobenthos has extended only to a distance of about 1 km from the outlet, i.e., the changes in vegetation have been largest in those areas that remain ice-free in winter. Similar trends were also discernible at Olkiluoto, but to a clearly smaller extent, which was due to the definitely weaker level of background eutrophy and nutrient concentrations in the Bothnian Sea, and the differing local hydrographical and biological factors prevailing in the Olkiluoto area. The level of primary production has also increased at Olkiluoto, but has remained at a clearly lower level than at Loviisa. In spite of the analogous changes observed in the macrozoobenthos, the benthic fauna has remained strong and diversified in the Olkiluoto area. Small amounts of local discharge nuclides were regularly detected in environmental samples taken from the discharge areas: tritium in seawater samples, and activation products, such as 60Co, 58Co, 54Mn, 110mAg, 51Cr, in suspended particulate matter, bottom sediments and in several indicator organisms (e.g., periphyton and Fucus vesiculosus) that effectively accumulate radioactive substances from the medium. The tritium discharges and the consequent detection frequency and concentrations of tritium in seawater were higher at Loviisa, but the concentrations of the activation products were higher at Olkiluoto, where traces of local discharge nuclides were also observed over a clearly wider area, due to the better exchange of water than at Loviisa, where local discharge nuclides were only detected outside Hästholmsfjärden Bay quite rarely and in smaller amounts. At the farthest, an insignificant trace amount (0.2 Bq kg-1 d.w.) of 60Co originating from Olkiluoto was detected in Fucus at a distance of 137 km from the power plant. Discharge nuclides from the local nuclear power plants were almost exclusively detected at the lower trophic levels of the ecosystems. Traces of local discharge nuclides were very seldom detected in fish, and even then only in very low quantities. As a consequence of the reduced discharges, the concentrations of local discharge nuclides in the environment have decreased noticeably in recent years at both Loviisa and Olkiluoto. Although the concentrations in environmental samples, and above all, the discharge data, are presented as seemingly large numbers, the radiation doses caused by them to the population and to the biota are very low, practically insignificant. The effects of the thermal discharges have been more significant, at least to the wildlife in the discharge areas of the cooling water, although the area of impact has been relatively small. The results show that the nutrient level and the exchange of water in the discharge area of a nuclear power plant are of crucial importance.

The Significance of Wild Plants in the Evolutionary Ecology of Three Major Viruses Infecting Cultivated Sweetpotato in Uganda

The studies presented in this thesis contribute to the understanding of evolutionary ecology of three major viruses threatening cultivated sweetpotato (Ipomoea batatas Lam) in East Africa: Sweet potato feathery mottle virus (SPFMV; genus Potyvirus; Potyviridae), Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus; Closteroviridae) and Sweet potato mild mottle virus (SPMMV; genus Ipomovirus; Potyviridae). The viruses were serologically detected and the positive results confirmed by RT-PCR and sequencing. SPFMV was detected in 24 wild plant species of family Convolvulacea (genera Ipomoea, Lepistemon and Hewittia), of which 19 species were new natural hosts for SPFMV. SPMMV and SPCSV were detected in wild plants belonging to 21 and 12 species (genera Ipomoea, Lepistemon and Hewittia), respectively, all of which were previously unknown to be natural hosts of these viruses. SPFMV was the most abundant virus being detected in 17% of the plants, while SPMMV and SPCSV were detected in 9.8% and 5.4% of the assessed plants, respectively. Wild plants in Uganda were infected with the East African (EA), common (C), and the ordinary (O) strains, or co-infected with the EA and the C strain of SPFMV. The viruses and virus-like diseases were more frequent in the eastern agro-ecological zone than the western and central zones, which contrasted with known incidences of these viruses in sweetpotato crops, except for northern zone where incidences were lowest in wild plants as in sweetpotato. The NIb/CP junction in SPMMV was determined experimentally which facilitated CP-based phylogenetic and evolutionary analyses of SPMMV. Isolates of all the three viruses from wild plants were genetically similar to those found in cultivated sweetpotatoes in East Africa. There was no evidence of host-driven population genetic structures suggesting frequent transmission of these viruses between their wild and cultivated hosts. The p22 RNA silencing suppressor-encoding sequence was absent in a few SPCSV isolates, but regardless of this, SPCSV isolates incited sweet potato virus disease (SPVD) in sweetpotato plants co-infected with SPFMV, indicating that p22 is redundant for synergism between SCSV and SPFMV. Molecular evolutionary analysis revealed that isolates of strain EA of SPFMV that is largely restricted geographically in East Africa experience frequent recombination in comparison to isolates of strain C that is globally distributed. Moreover, non-homologous recombination events between strains EA and C were rare, despite frequent co-infections of these strains in wild plants, suggesting purifying selection against non-homologous recombinants between these strains or that such recombinants are mostly not infectious. Recombination was detected also in the 5 - and 3 -proximal regions of the SPMMV genome providing the first evidence of recombination in genus Ipomovirus, but no recombination events were detected in the characterized genomic regions of SPCSV. Strong purifying selection was implicated on evolution of majority of amino acids of the proteins encoded by the analyzed genomic regions of SPFMV, SPMMV and SPCSV. However, positive selection was predicted on 17 amino acids distributed over the whole the coat protein (CP) in the globally distributed strain C, as compared to only 4 amino acids in the multifunctional CP N-terminus (CP-NT) of strain EA largely restricted geographically to East Africa. A few amino acid sites in the N-terminus of SPMMV P1, the p7 protein and RNA silencing suppressor proteins p22 and RNase3 of SPCSV were also submitted to positive selection. Positively selected amino acids may constitute ligand-binding domains that determine interactions with plant host and/or insect vector factors. The P1 proteinase of SPMMV (genus Ipomovirus) seems to respond to needs of adaptation, which was not observed with the helper component proteinase (HC-Pro) of SPMMV, although the HC-Pro is responsible for many important molecular interactions in genus Potyvirus. Because the centre of origin of cultivated sweetpotato is in the Americas from where the crop was dispersed to other continents in recent history (except for the Australasia and South Pacific region), it would be expected that identical viruses and their strains occur worldwide, presuming virus dispersal with the host. Apparently, this seems not to be the case with SPMMV, the strain EA of SPFMV and the strain EA of SPCSV that are largely geographically confined in East Africa where they are predominant and occur both in natural and agro-ecosystems. The geographical distribution of plant viruses is constrained more by virus-vector relations than by virus-host interactions, which in accordance of the wide range of natural host species and the geographical confinement to East Africa suggest that these viruses existed in East African wild plants before the introduction of sweetpotato. Subsequently, these studies provide compelling evidence that East Africa constitutes a cradle of SPFMV strain EA, SPCSV strain EA, and SPMMV. Therefore, sweet potato virus disease (SPVD) in East Africa may be one of the examples of damaging virus diseases resulting from exchange of viruses between introduced crops and indigenous wild plant species. Keywords: Convolvulaceae, East Africa, epidemiology, evolution, genetic variability, Ipomoea, recombination, SPCSV, SPFMV, SPMMV, selection pressure, sweetpotato, wild plant species Author s Address: Arthur K. Tugume, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Latokartanonkaari 7, P.O Box 27, FIN-00014, Helsinki, Finland. Email: tugume.arthur@helsinki.fi Author s Present Address: Arthur K. Tugume, Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Kampala, Uganda. Email: aktugume@botany.mak.ac.ug, tugumeka@yahoo.com

Overwintering ecology of northern field layer plants : snow and photosynthesis in Vaccinium vitis-idaea L.

Winter is a significant period for the seasonality of northern plants, but is often overlooked when studying the interactions of plants and their environment. This study focuses on the effects of overwintering conditions, including warm winter periods, snow, and snowmelt on boreal and sub-Arctic field layer plants. Wintertime photosynthesis and related physiological factors of evergreen dwarf shrubs, particularly of Vaccinium vitis-idaea, are emphasised. The work combines experiments both in the field and in growth chambers with measurements in natural field conditions. Evergreen dwarf shrubs are predominantly covered by snow in the winter. The protective snow cover provides favourable conditions for photosynthesis, especially during the spring before snowmelt. The results of this study indicate that photosynthesis occurs under the snow in V. vitis-idaea. The light response of photosynthesis determined in field conditions during the period of snow cover shows that positive net CO2 exchange is possible under the snow in the prevailing light and temperature. Photosynthetic capacity increases readily during warm periods in winter and the plants are thus able to replenish carbohydrate reserves lost through respiration. Exposure to low temperatures in combination with high light following early snowmelt can set back photosynthesis as sustained photoprotective measures are activated and photodamage begins to build up. Freezing may further decrease the photosynthetic capacity. The small-scale distribution of many field layer plants, including V. vitis-idaea and other dwarf shrubs, correlates with the snow distribution in a forest. The results of this study indicate that there are species-specific differences in the snow depth affinity of the field and ground layer species. Events and processes taking place in winter can have a profound effect on the overall performance of plants and on the interactions between plants and their environment. Understanding the processes involved in the overwintering of plants is increasingly important as the wintertime climate in the north is predicted to change in the future.

Dracocephalum moldavica L. and Melissa officinalis L. : Chemistry and Bioactivities Relevant in Alzheimer’s Disease Therapy

Oksidatiivisen stressin eli liiallisen reaktiivisten happiyhdisteiden määrän soluissa on jo pitkään arveltu olevan tärkeä Alzheimerin taudin kehittymiseen ja etenemiseen vaikuttava tekijä. Tämän vuoksi kiinnostus erilaisten antioksidanttien (yhdisteitä, jotka neutraloivat näitä happiradikaaleja soluissa) mahdollisia terapeuttisia ominaisuuksia Alzheimerin taudin hoidossa on tutkittu laajalti. Tähän mennessä ei kuitenkaan ole vielä onnistuttu löytämään antioksidanttia, joka olisi hyödyksi Alzheimerin taudin hoidossa. Tämän vuoksi on tärkeää pyrkiä löytämään uusia antioksidanttien lähteitä sekä tutkia niistä löytyviä aktiivisia yhdisteitä. Kiinnostus luonnon antioksidantteja kohtaan on kasvanut voimakkaasti viime aikoina. Huomio on kiinnittynyt erityisesti aromaattisista sekä lääkekasveista löytyviin antioksidantteihin. Lamiaceae- perheeseen kuuluvia tuoksuampiaisyrttiä (Dracocephalum moldavica L.) ja sitruunamelissaa (Melissa officinalis L.) on käytetty Iranissa pitkään sekä ruoanlaitossa että lääkinnässä, minkä vuoksi näiden kasvien uutteiden antioksidanttisisältöä päätettiin analysoida käyttäen useaa erilaista in vitro- menetelmää. Näissä kokeissa ilmeni, että uutteilla oli useita antioksidanttisia vaikutuksia. Näistä antioksidanttisista vaikutuksista vastaavia yhdisteitä pyrittiin tunnistamaan käyttäen HPLC-PDA- tekniikkaa, minkä seurauksena niiden havaittiin sisältävän erilaisia polyfenoleita, kuten hydroksyloituneita bentsoeeni- ja cinnamamidihapon johdannaisia sekä flavonoideja. Kummankin kasvin uutteissa runsaimmin esiintynyt yhdiste oli rosmariinihappo. Sitruunamelissaa (M. officinalis) on käytetty antiikin ajoista alkaen kognitiivisten toimintojen häiriöiden hoidossa. Perustuen tietoon kasvin käytöstä perinteisessä lääkinnässä, sen tehoa Alzheimerin taudin hoidossa on tutkittu viime aikoina kliinisin kokein. Sitruunamelissan todettiinkin olevan hyödyksi lievää ja keskivaikeaa Alzheimeimerin tautia sairastavien potilaiden hoidossa. Väitöskirjan osanan olevasta kooste-artikkelista käy ilmi, että tutkimalla lääkekasvien ominaisuuksia voidaan saada arvokkaita suuntaa-antavia vihjeitä Alzheimerin taudin lääkehoidon kehittämiseen. Tämän perusteella päätettiinkin testatata myös sitruunamelissauutteen kykyä estää asetyylikoliiniesteraasin (AChE) toimintaa, koska tämän entsyymin toiminna estämisen tiedetään olevan hyödyksi Alzheimerin taudin hoidossa. Uute kykeni estämään AChE:n toimintaa, minkä vuoksi uutteen sisältämiä komponentteja päätettiin tutkia terkemmin. Uute jaettiin erilaisiin fraktioihin käyttäen HPLC-menetelmää, minkä jälkeen testattiin jokaisen fraktion kykyä inhiboida AchE. Suurin osa fraktioista kykeni inhiboimaan AChE:n toimintaa selkeästi tehokkaammin, kuin raakauute. Kaikista tehokkainta fraktiota analysoitiin tarkemmin sen aktiivisten yhdisteiden tunnistamiseksi, minkä seurauksena sen sisältämät yhdisteet tunnistettiin cis ja trans-rosmariinihapoiksi. Tässä tutkimuksessa tunnistettujen yhdisteiden hyödyllisyyttä Alzheimerin taudin hoidossa tulisi seuraavaksi tutkia erilaisissa in vivo-malleissa. Lisäksi jäljellä olevien fraktioiden kemiallinen koostumus tulisi selvittää sekä antioksidanttiaktiivisuuden ja AChE:n toiminnan inhiboinnin välistä mahdollista yhteyttä tulisi tutkia tarkemmin. Tämä tutkimus osoittaa tuoksuampiasyrtin (D. moldavica) sekä sitruunamelissan (M. officinalis) sisältävän monenlaisia aktiivisia antioksidantteja. Lisäksi sitruunamelissan sisältämät yhdisteet kykenivät estämään asetyylikoliiniesteraasin (AchE) toimintaa. Nämä tulokset tukevat osaltaan väitöskirjan osana olevan kooste-artikkelin johtopäätöksiä, joiden mukaan etnofarmakologinen kasvitutkimus voi osoittautua erittäin hyödylliseksi kehitettäessä uutta lääkehoitoa Alzheimerin tautiin. Lisäksi tässä väitöskirjassa kuvattu tutkimus osoittaakin, että perinteisesti lääkekasvina käytettyä sitruunamelissaa voidaan mahdollisesti hyödyntää uusien Alzheimerin taudin hoitoon käytettävien lääkkeiden kehityksessä.

Heterotrophic Bacteria Associated with Cyanobacteria in Recreational and Drinking Water

Cyanobacterial mass occurrences, also known as water blooms, have been associated with adverse health effects of both humans and animals. They can also be a burden to drinking water treatment facilities. Risk assessments of the blooms have generally focused on the cyanobacteria themselves and their toxins. However, heterotrophic bacteria thriving among cyanobacteria may also be responsible for many of the adverse health effects, but their role as the etiological agents of these health problems is poorly known. In addition, studies on the water purification efficiency of operating water treatment plants during cyanobacterial mass occurrences in their water sources are rare. In the present study, over 600 heterotrophic bacterial strains were isolated from natural freshwater, brackish water or from treated drinking water. The sampling sites were selected as having frequent cyanobacterial occurrences in the water bodies or in the water sources of the drinking water treatment plants. In addition, samples were taken from sites where cyanobacterial water blooms were surmised to have caused human health problems. The isolated strains represented bacteria from 57 different genera of the Gamma-, Alpha- or Betaproteobacteria, Actinobacteria, Flavobacteria, Sphingobacteria, Bacilli and Deinococci classes, based on their partial 16S rRNA sequences. Several isolates had no close relatives among previously isolated bacteria or cloned 16S rRNA genes of uncultivated bacteria. The results show that water blooms are associated with a diverse community of cultivable heterotrophic bacteria. Chosen subsets of the isolated strains were analysed for features such as their virulence gene content and possible effect on cyanobacterial growth. Of the putatively pathogenic haemolytic strains isolated in the study, the majority represented the genus Aeromonas. Therefore, the Aeromonas spp. strains isolated from water samples associated with adverse health effects were screened for the virulence gene types encoding for enterotoxins (ast, alt and act/aerA/hlyA), flagellin subunits (flaA/flaB), lipase (lip/pla/lipH3/alp-1) and elastase (ahyB) by PCR. The majority (90%) of the Aeromonas strains included one or more of the six screened Aeromonas virulence gene types. The most common gene type was act, which was present in 77% of the strains. The fla, ahyB and lip genes were present in 30 37% of the strains. The prevalence of the virulence genes implies that the Aeromonas may be a factor in some of the cyanobacterial associated health problems. Of the 183 isolated bacterial strains that were studied for possible effects on cyanobacterial growth, the majority (60%) either enhanced or inhibited growth of cyanobacteria. In most cases, they enhanced the growth, which implies mutualistic interactions. The results indicate that the heterotrophic bacteria have a role in the rise and fall of the cyanobacterial water blooms. The genetic and phenotypic characteristics and the ability to degrade cyanobacterial hepatotoxins of 13 previously isolated Betaproteobacteria strains, were also studied. The strains originated from Finnish lakes with frequent cyanobacterial occurrence. Tested strains degraded microcystins -LR and -YR and nodularin. The strains could not be assigned to any described bacterial genus or species based on their genetic or phenotypic features. On the basis of their characteristics a new genus and species Paucibacter toxinivorans was proposed for them. The water purification efficiency of the drinking water treatment processes during cyanobacterial water bloom in water source was assessed at an operating surface water treatment plant. Large phytoplankton, cyanobacterial hepatotoxins, endotoxins and cultivable heterotrophic bacteria were efficiently reduced to low concentrations, often below the detection limits. In contrast, small planktonic cells, including also possible bacterial cells, regularly passed though the water treatment. The passing cells may contribute to biofilm formation within the water distribution system, and therefore lower the obtained drinking water quality. The bacterial strains of this study offer a rich source of isolated strains for examining interactions between cyanobacteria and the heterotrophic bacteria associated with them. The degraders of cyanobacterial hepatotoxins could perhaps be utilized to assist the removal of the hepatotoxins during water treatment, whereas inhibitors of cyanobacterial growth might be useful in controlling cyanobacterial water blooms. The putative pathogenicity of the strains suggests that the health risk assessment of the cyanobacterial blooms should also cover the heterotrophic bacteria.

Urban energy fluxes in Helsinki and their high frequency spectral corrections

Inadvertent climate modification has led to an increase in urban temperatures compared to the surrounding rural area. The main reason for the temperature rise is the altered energy portioning of input net radiation to heat storage and sensible and latent heat fluxes in addition to the anthropogenic heat flux. The heat storage flux and anthropogenic heat flux have not yet been determined for Helsinki and they are not directly measurable. To the contrary, turbulent fluxes of sensible and latent heat in addition to net radiation can be measured, and the anthropogenic heat flux together with the heat storage flux can be solved as a residual. As a result, all inaccuracies in the determination of the energy balance components propagate to the residual term and special attention must be paid to the accurate determination of the components. One cause of error in the turbulent fluxes is the fluctuation attenuation at high frequencies which can be accounted for by high frequency spectral corrections. The aim of this study is twofold: to assess the relevance of high frequency corrections to water vapor fluxes and to assess the temporal variation of the energy fluxes. Turbulent fluxes of sensible and latent heat have been measured at SMEAR III station, Helsinki, since December 2005 using the eddy covariance technique. In addition, net radiation measurements have been ongoing since July 2007. The used calculation methods in this study consist of widely accepted eddy covariance data post processing methods in addition to Fourier and wavelet analysis. The high frequency spectral correction using the traditional transfer function method is highly dependent on relative humidity and has an 11% effect on the latent heat flux. This method is based on an assumption of spectral similarity which is shown not to be valid. A new correction method using wavelet analysis is thus initialized and it seems to account for the high frequency variation deficit. Anyhow, the resulting wavelet correction remains minimal in contrast to the traditional transfer function correction. The energy fluxes exhibit a behavior characteristic for urban environments: the energy input is channeled to sensible heat as latent heat flux is restricted by water availability. The monthly mean residual of the energy balance ranges from 30 Wm-2 in summer to -35 Wm-2 in winter meaning a heat storage to the ground during summer. Furthermore, the anthropogenic heat flux is approximated to be 50 Wm-2 during winter when residential heating is important.

Role of Temperature in the Biological Activity of a Boreal Forest

In northern latitudes, temperature is the key factor driving the temporal scales of biological activity, namely the length of the growing season and the seasonal efficiency of photosynthesis. The formation of atmospheric concentrations of biogenic volatile organic compounds (BVOCs) are linked to the intensity of biological activity. However, interdisciplinary knowledge of the role of temperature in the biological processes related to the annual cycle and photosynthesis and atmospheric chemistry is not fully understood. The aim of this study was to improve understanding of the role of temperature in these three interlinked areas: 1) onset of growing season, 2) photosynthetic efficiency and 3) BVOC air concentrations in a boreal forest. The results present a cross-section of the role of temperature on different spatial (southern northern boreal), structural (tree forest stand - forest) and temporal (day-season- year) scales. The fundamental status of the Thermal Time model in predicting the onset of spring recovery was confirmed. However, it was recommended that sequential models would be more appropriate tools when the onset of the growing season is estimated under a warmer climate. A similar type of relationship between photosynthetic efficiency and temperature history was found in both southern and northern boreal forest stands. This result draws attention to the critical question of the seasonal efficiency of coniferous species to emit organic compounds under a warmer climate. New knowledge about the temperature dependence of the concentrations of biogenic volatile organic compounds in a boreal forest stand was obtained. The seasonal progress and the inter-correlation of BVOC concentrations in ambient air indicated a link to biological activity. Temperature was found to be the main driving factor for the concentrations. However, in addition to temperature, other factors may play a significant role here, especially when the peak concentrations are studied. There is strong evidence that the spring recovery and phenological events of many plant species have already advanced in Europe. This study does not fully support this observation. In a boreal forest, changes in the annual cycle, especially the temperature requirement in winter, would have an impact on the atmospheric BVOC composition. According to this study, more joint phenological and BVOC field observations and laboratory experiments are still needed to improve these scenarios.

Archaea, Bacteria, and methane production along environmental gradients in fens and bogs

Metanogeenit ovat hapettomissa oloissa eläviä arkkien pääryhmään kuuluvia mikrobeja, joiden ainutlaatuisen aineenvaihdunnan seurauksena syntyy metaania. Ilmakehässä metaani on voimakas kasvihuonekaasu. Yksi suurimmista luonnon metaanilähteistä ovat kosteikot. Pohjoisten soiden metaanipäästöt vaihtelevat voimakkaasti eri soiden välillä ja yhden suon sisälläkin, riippuen muun muassa vuodenajasta, suotyypistä ja kasvillisuudesta. Väitöskirjatyössä tutkittiin metaanipäästöjen vaihtelun mikrobiologista taustaa. Tutkimuksessa selvitettiin suotyypin, vuodenajan, tuhkalannoituksen ja turvesyvyyden vaikutusta metanogeeniyhteisöihin sekä metaanintuottoon kolmella suomalaisella suolla. Lisäksi tutkittiin ei-metanogeenisia arkkeja ja bakteereita, koska ne muodostavat metaanin tuoton lähtöaineet osana hapetonta hajotusta. Mikrobiyhteisöt analysoitiin DNA- ja RNA-lähtöisillä, polymeraasiketjureaktioon (PCR) perustuvilla menetelmillä. Merkkigeeneinä käytettiin metaanin tuottoon liittyvää mcrA-geeniä sekä arkkien ja bakteerien ribosomaalista 16S RNA-geeniä. Metanogeeniyhteisöt ja metaanintuotto erosivat huomattavasti happaman ja vähäravinteisen rahkasuon sekä ravinteikkaampien sarasoiden välillä. Rahkasuolta löytyi lähes yksinomaan Methanomicrobiales-lahkon metanogeeneja, jotka tuottavat metaania vedystä ja hiilidioksidista. Sarasoiden metanogeeniyhteisöt olivat monimuotoisempia, ja niillä esiintyi myös asetaattia käyttäviä metanogeeneja. Vuodenaika vaikutti merkittävästi metaanintuottoon. Talvella havaittiin odottamattoman suuri metaanintuottopotentiaali sekä viitteitä aktiivisista metanogeeneista. Arkkiyhteisön koostumus sen sijaan vaihteli vain vähän. Tuhkalannoitus, jonka tarkoituksena on edistää puiden kasvua ojitetuilla soilla, ei merkittävästi vaikuttanut metaanintuottoon tai -tuottajiin. Ojitetun suon yhteisöt kuitenkin muuttuivat turvesyvyyden mukaan. Vertailtaessa erilaisia PCR-menetelmiä todettiin, että kolmella mcrA-geeniin kohdistuvalla alukeparilla havaittiin pääosin samat ojitetun suon metanogeenit, mutta lajien runsaussuhteet riippuvat käytetyistä alukkeista. Soilla havaitut bakteerit kuuluivat pääjaksoihin Deltaproteobacteria, Acidobacteria ja Verrucomicrobia. Lisäksi löydettiin Crenarchaeota-pääjakson ryhmiin 1.1c ja 1.3 kuuluvia ei-metanogeenisia arkkeja. Tulokset ryhmien esiintymisestä hapettomassa turpeessa antavat lähtökohdan selvittää niiden mahdollisia vuorovaikutuksia metanogeenien kanssa. Tutkimuksen tulokset osoittivat, että metanogeeniyhteisön koostumus heijastaa metaanintuottoon vaikuttavia kemiallisia tai kasvillisuuden vaihteluita kuten suotyyppiä. Soiden metanogeenien ja niiden fysiologian parempi tuntemus voi auttaa ennustamaan ympäristömuutosten vaikutusta soiden metaanipäästöihin.

Archaea in the Mycorrhizosphere of Boreal Forest Trees

Archaea were long thought to be a group of ancient bacteria, which mainly lived in extreme environments. Due to the development of DNA sequencing methods and molecular phylogenetic analyses, it was shown that the living organisms are in fact divided into three domains; the Archaea, Bacteria and the Eucarya. Since the beginning of the previous decade, it was shown that archaea generally inhabit moderate environments and that these non-extremophilic archaea are more ubiquitous than the extremophiles. Group 1 of non-extreme archaea affiliate with the phylum Crenarchaeota. The most commonly found soil archaea belong to the subgroup 1.1b. However, the Crenarchaeota found in the Fennoscandian boreal forest soil belong to the subgroup 1.1c. The organic top layer of the boreal forest soil, the humus, is dominated by ectomycorrhizal fungal hyphae. These colonise virtually all tree fine root tips in the humus layer and have been shown to harbour distinct bacterial populations different from those in the humus. The archaea have also been shown to colonise both boreal forest humus and the rhizospheres of plants. In this work, studies on the archaeal communities in the ectomycorrhizospheres of boreal forest trees were conducted in microcosms. Archaea belonging to the group 1.1c Crenarchaeota and Euryarchaeota of the genera Halobacterium and Methanolobus were detected. The archaea generally colonised fungal habitats, such as ectomycorrhizas and external mycelia, rather than the non-mycorrhizal fine roots of trees. The species of ectomycorrhizal fungus had a great impact on the archaeal community composition. A stable euryarchaeotal community was detected especially in the mycorrhizas, of most of the tested Scots pine colonising ectomycorrhizal fungi. The Crenarchaeota appeared more sporadically in these habitats, but had a greater diversity than the Euryarchaeota. P. involutus mycorrhizas had a higher diversity of 1.1c Crenarchaeota than the other ectomycorrhizal fungi. The detection level of archaea in the roots of boreal trees was generally low although archaea have been shown to associate with roots of different plants. However, alder showed a high diversity of 1.1c Crenarchaeota, exceeding that of any of the tested mycorrhizas. The archaeal 16S rRNA genes detected from the non-mycorrhizal roots were different from those of the P. involutus mycorrhizas. In the phylogenetic analyses, the archaeal 16S rRNA gene sequences obtained from non-mycorrhizal fine roots fell in a separate cluster within the group 1.1c Crenarchaeota than those from the mycorrhizas. When the roots of the differrent tree species were colonised by P. involutus, the diversity and frequency of the archaeal populations of the different tree species were more similar to each other. Both Cren- and Euryarchaeota were enriched in cultures to which C-1 substrates were added. The 1.1c Crenarchaeota grew anaerobically in mineral medium with CH4 and CO2 as the only available C sources, and in yeast extract media with CO2 and CH4 or H2. The crenarchaeotal diversity was higher in aerobic cultures on mineral medium with CH4 or CH3OH than in the anaerobic cultures. Ecological functions of the mycorrhizal 1.1c Crenarchaeota in both anaerobic and aerobic cycling of C-1 compounds were indicated. The phylogenetic analyses did not divide the detected Crenarchaeota into anaerobic and aerobic groups. This may suggest that the mycorrhizospheric crenarchaeotal communities consist of closely related groups of anaerobic and aerobic 1.1c Crenarchaeota, or the 1.1c Crenarchaeota may be facultatively anaerobic. Halobacteria were enriched in non-saline anaerobic yeast extract medium cultures in which CH4 was either added or produced, but were not detected in the aerobic cultures. They may potentially be involved in anaerobic CH4 cycling in ectomycorrhizas. The CH4 production of the mycorrhizal samples was over 10 times higher than for humus devoid of mycorrhizal hyphae, indicating a high CH4 production potential of the mycorrhizal metanogenic community. Autofluorescent methanogenic archaea were detected by microscopy and 16S rRNA gene sequences of the genus Methanolobus were obtained. The archaeal community depended on both tree species and the type of ectomycorrhizal fungus colonising the roots and the Cren- and Euryarchaeota may have different ecological functions in the different parts of the boreal forest tree rhizosphere and mycorrhizosphere. By employing the results of this study, it may be possible to isolate both 1.1c Crenarchaeota as well as non-halophilic halobacteria and aerotolerant methanogens from mycorrhizospheres. These archaea may be used as indicators for change in the boreal forest soil ecosystem due to different factors, such as exploitations of forests and the rise in global temperature. More information about the microbial populations with apparently low cell numbers but significant ecological impacts, such as the boreal forest soil methanogens, may be of crucial importance to counteract human impacts on such globally important ecosystems as the boreal forests.

The ecology of the mountain hare (Lepus timidus L.) in managed boreal forests: habitat associations at different forest scales

Aims of this thesis This study is part of a larger hare project in Finland, which provides answers to basic ecological questions regarding the mountain hare. This study of the ecology of the mountain hare focuses in particular on different levels of managed boreal forest. The feeding habits and intensity of mountain hares in winter are explored, and the connections between mountain hares versus the forest structure are also studied (e.g. habitat use and the importance of different forest layers for hares). The use of the environment by hares at the landscape level was examined (forest patch structures), and the home ranges of mountain hares were studied. Finally, the productivity and survival rate of mountain hare populations were also studied (discussion e.g. predator effects on hare populations). Conclusions Feeding intensity seemed to be highest in the spring-winter, when home ranges were also largest. Favourable food species are covered by snow in winter and the mobility of hares is highest during late winter. A shortage of suitable food species may be problematic for hares, especially during the winter period. In this study mountain hares preferred a dense shrub layer at local level and deciduous and mixed tree forest over coniferous forest at the landscape level. Food and shelter are vital for hares and the preference for particular habitats may also affect the population dynamics of the mountain hare. It would be possible to improve the quality of food and shelter or at least prevent the most negative habitat changes through forest management. At a local level it is also possible to add supplementary food for hares through the winter period. The intensive clearing of young sapling stands and especially the removal of deciduous shrubs and trees reduces the quality of habitats for the mountain hare. Mountain hares primarily live in forest habitat and it is possible that changes in the forest structure play a crucial role in mountain hare habitat preference. Ecological knowledge of the mountain hare is vital to create habitat structure more suitable for the species. More deciduous trees should be saved in managing forests and the mechanical clearing of the shrub layer should be done carefully.

Lignin biosynthesis in Norway spruce: from a model system to the tree

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.

Secondary metabolites in Gerbera hybrida

Plants produce a diversity of secondary metabolites, i.e., low-molecular-weight compounds that have primarily ecological functions in plants. The flavonoid pathway is one of the most studied biosynthetic pathways in plants. In order to understand biosynthetic pathways fully, it is necessary to isolate and purify the enzymes of the pathways to study individual steps and to study the regulatory genes of the pathways. Chalcone synthases are key enzymes in the formation of several groups of flavonoids, including anthocyanins. In this study, a new chalcone synthase enzyme (GCHS4), which may be one of the main contributors to flower colour, was characterised from the ornamental plant Gerbera hybrida. In addition, four chalcone synthase-like genes and enzymes (GCHS17, GCHS17b, GCHS26 and GCHS26b) were studied. Spatial expression of the polyketide synthase gene family in gerbera was also analysed with quantitative RT-PCR from 12 tissues, including several developmental stages and flower types. A previously identified MYB transcription factor from gerbera, GMYB10, which regulates the anthocyanin pathway, was transferred to gerbera and the phenotypes were analysed. Total anthocyanin content and anthocyanidin profiles of control and transgenic samples were compared spectrophotometrically and with HPLC. The overexpression of GMYB10 alone was able to change anthocyanin pigmentation: cyanidin pigmentation was induced and pelargonidin pigmentation was increased. The gerbera 9K cDNA microarray was used to compare the gene expression profiles of transgenic tissues against the corresponding control tissues to reveal putative target genes for GMYB10. GMYB10 overexpression affected the expression of both early and late biosynthetic genes in anthocyanin-accumulating transgenic tissues, including the newly isolated gene GCHS4. Two new MYB domain factors, named as GMYB11 and GMYB12, were also upregulated. Gene transfer is not only a powerful tool for basic research, but also for plant breeding. However, crop improvement by genetic modification (GM) remains controversial, at least in Europe. Many of the concerns relating to both human health and to ecological impacts relate to changes in the secondary metabolites of GM crops. In the second part of this study, qualitative and quantitative differences in cytotoxicity and metabolic fingerprints between 225 genetically modified Gerbera hybrida lines and 42 non-GM Gerbera varieties were compared. There was no evidence for any major qualitative and quantitative changes between the GM lines and non-GM varieties. The developed cell viability assays offer also a model scheme for cell-based cytotoxicity screening of a large variety of GM plants in standardized conditions.

Identification and Epidemiological Typing of Campylobacter strains isolated from Patients in Finland

C. jejuni constitutes the majority of Campylobacter strains isolated from patients in Finland, and C. coli strains are also reported. To improve the species identification, a combination of phenotype- and genotype-based methods was applied. Standardising the cell suspension turbidity in the hippurate hydrolysis test enabled the reliable identification of hippurate-positive Campylobacter strains as C. jejuni. The detection of species-specific genes by PCR showed that about 30% of the hippurate-negative strains were C. jejuni. Three typing methods, serotyping, PCR-RFLP analysis of LOS biosynthesis genes and pulsed-field gel electrophoresis (PFGE) were evaluated as epidemiological typing tools for C. jejuni. The high number of non-typeable strains lowered the discriminatory ability of serotyping. PCR-RFLP typing offered high discrimination for both serotypeable and non-typeable strains, but the correlation between serotypes and RFLP-types was not high enough to enable its use for molecular serotyping of non-typeable strains. PFGE was a highly discriminative typing method. Although the use of two restriction enzymes generally increases the discriminatory ability, KpnI alone offered almost as high discrimination as the use of SmaI and KpnI. The characteristic seasonal distribution of Campylobacter infections with a peak in summer and low incidence in winter was mainly due to domestically acquired infections. Of the C. jejuni strains, 41% were of domestic origin compared to only 17% of the C. coli strains. Serotypes Pen 12, Pen 6,7 and Pen 27 were significantly associated with domestic C. jejuni infections, Pen 1,44, Pen 3 and Pen 37 with travel-related infections. Pen 2 and Pen 4-complex were common both in domestic and travel-related infections. Serotype Pen 2 was less common among patients 60 years or older than in younger patients, more prevalent in Western Finland than in other parts of the country and more prevalent than other serotypes in winter. The source of Pen 2 infections may be related to cattle, since Pen 2 is the most common serotype in isolates from Finnish cattle. PFGE subtypes among isolates from patients and chickens during the summer 2003 and from cattle during the whole year were compared. The analysis of indistinguishable SmaI/KpnI subtypes suggested that up to 31% of the human infections may have been mediated by chickens and 19% by cattle. Human strains isolated during two one-year sampling periods were studied by PFGE. Of the domestic strains, 69% belonged to SmaI subtypes found during both sampling periods. Four SmaI subtypes accounted for 45% of the domestic strains, further typing of these subtypes by KpnI revealed six temporally persistent SmaI/KpnI subtypes. They were only occasionally identified in travel-related strains, and therefore, can be considered to be national subtypes. Each subtype was associated with a serotype: Pen 2, Pen 12, Pen 27, Pen 4-complex, Pen 41, and Pen 57. Five of these subtypes were identified in cattle (S5/K27, S7/K1, S7/K2, S7/K5 and S64/K19), and two in chickens (S7/K1 and S64/K19) with a temporal association with human infections in 2003. Cattle are more likely potential sources of these persistent subtypes, since long-term excretion of Campylobacter strains by cattle has been reported.