BIOLOGICAL INTERACTIONS GRADUATE SCHOOL
ANNUAL SEMINAR
10-11.11. 2003
Caribia Hotel, Turku
PROGRAM:
MONDAY 10.11.
12-13.00
Lunch
13-14.10
Opening words, Introductions and Session I
Chair Saija Pursiheimo
Marianne Mäntymäki: Hypoxia-inducible factors
Eeva Rissanen: Oxygen-dependent gene expression in trout hepatocytes
Kristiina Vuori: Differential gene expression in Baltic salmon yolk-sac fry
suffering from early mortality syndrome (M74) analyzed using suppression
subtractive hybridisation (SSH) and cDNA microarray
14.10-14.20
Break
14.20-16.00
“Biologist in public”
Markku Lappalainen: “Biologi julkisuudessa”
Mikko Nikinmaa: “Publicity from a scientist’s standpoint”
Discussions on scientific presentation
Coffee
16 -17.20
Session II
Chair Mari Nykänen
Marjaana Suorsa: Protein assembly of photosystem II and accumulation of
subcomplexes in the absence of low molecular weight subunits PsbL and PsbJ
Tove Jansen: Post-genomic research of Synechocystis 6803 ctp gene family
Mirva Piippo: Photosynthetic redox regulation of Arabidopsis nuclear genes
Pengpeng Zhang: Novel in sights into cyanobacterial membranes using
proteomics approach
17.3020.30-
Plop in the Spa
Dinner
TUESDAY 11.11
7-9.00
9-10.20
Morning swimming and Breakfast
Session III
Chair Olli Ylönen
Lotta-Riina Suomalainen: Genotypic analysis of Flavobacterium columnare
isolates in Finland
Anssi Karvonen: Parasite avoidance behaviour in preventing eye fluke
infections in fish
Otto Seppälä: Altered behaviour and predation vulnerability of fish infected
with trophically transmitted parasites
Anssi Vainikka: The multiple applications of the immunocompetencehandicap hypothesis
10.20-10.30
Break
10.30-11.50 Session IV
Chair Mirkka Herranen
Mikko Olin: The effects of biomanipulation – study of ten eutrophic lakes in
southern Finland
Mari Nykänen: Spawning habitat requirements of riverine European rgayling
Thymallus thymallus L.
Marianna Riipi: Temporal correlations in the leaf chemistry of mountain
birch: are the same trees always resistant to herbivores?
Lauri Kapari: Shading induced variability in mountain birch leaf chemistry –
effects on performance and faecal composition of sawflies
12-13
Lunch
13-
Session V
Saijaliisa Pursiheimo
Soile Jokipii: Heterologous hemoglobin (vhb)-gene effects on pathogen
resistance and mycorrhizal interaction in hybrid aspen (Populus tremula x
tremuloides)
Minna Hassinen: Importance of cardiac potassium channels in temperature
acclimation of rainbow trout heart
Piia Leskinen: Developing yeast cell-based assays for measuring endocrine
disrupters in environment
Sari Myllymäki: The expression of steroidogenic enzymes in the developing
ovary of control and in utero TCDD-exposed Spraque-Dawley rat
Closing
HYPOXIA INDUCIBLE FACTOR-1 IN DIFFERENT FISH SPECIES
Marianne Mäntymäki and Mikko Nikinmaa
Laboratory of Animal Physiology, Department of Biology, University of Turku, FIN-20014
Turku, FINLAND
HIF-1 is a heterodimeric protein composed of the subunits HIF-1α and ARNT (HIF-1β), of
which the HIF-1α subunit confers the hypoxia sensitivity. HIF-1α is rapidly degraded by a
proteasome under normoxic conditions, mainly as a result of prolyl hydroxylation needed for
protein destabilization. Although prolyl hydroxylation at conserved proline residues is a major
factor controlling HIF-1α stability, several studies have shown that, in addition, the redox
state of the cells influences HIF-1α function, both its stability and phosphorylation.
Sensitivity of the protein to oxidation/reduction may be due to cysteine residues at critical
positions. The predicted amino acid sequence of rainbow trout HIF-1α contains several
unique cysteine residues in the transactivation domain of the molecule. If this were the case,
then environmental disturbances involving oxidative and reductive stresses would affect the
hypoxia responsive gene expression especially in salmonids. This could be the case for
several organic pollutants and heavy metals with variable valency state. The aim of the
present study is to clone HIF-1α from different Baltic fish species, research their HIF-1α
redox sensitivity. The aim of the study is also to do phylogenetic analyse using cloned HIF-1α
sequences to obtain new information about fish molecular evolution.
OXYGEN-DEPENDENT GENE EXPRESSION IN TROUT HEPATOCYTES
Eeva Rissanen
Laboratory of Animal Physiology, Department of Biology, University of Turku, FIN-20014
Turku, FINLAND
DIFFERENTIAL GENE EXPRESSION IN BALTIC SALMON YOLK-SAC FRY
SUFFERING FROM EARLY MORTALITY SYNDROME (M74) ANALYZED USING
SUPPRESSION SUBTRACTIVE HYBRIDISATION (SSH) AND cDNA
MICROARRAY
Kristiina A.M. Vuori*, Heikki Koskinen†, Aleksei Krasnov†, Heli Teerijoki‡, Pekka
Vuorinen§ and Mikko Nikinmaa*
*Laboratory of Animal Physiology, Department of Biology, University of Turku, FIN-20014
Turku, FINLAND
†Institute of Applied Biotechnology, University of Kuopio, P.O.B. 1627, Kuopio 70211,
FINLAND
‡ Present address: Turku Centre for Biotechnology, P.O.Box 123, BioCity, Turku, FIN20521,
FINLAND
§ Finnish Game and Fisheries Research Institute, PO Box 6, FIN-00721 Helsinki, FINLAND
Baltic salmon suffer from abnormally high, maternally transmitted yolk-sac fry mortality. In
1990´s 50-90% of newly hatched salmon from wild parents in Sweden and Finland died
before feeding. M74-affected fry are characterized by a low thiamine content and oxidative
stress. At the morphological level diseased fry appear to have e.g. impaired vascular
development, hemorrhages and reduced number of circulating erythrocytes, hepatocellular
lipid accumulation, dilatation of hepatocytic endoplasmic reticulum and degeneration of
skeletal muscle. Necrotic cells are observed in the brain. At the behavioural level, various
neurological disorders such as lack of light sensitivity, passivity and spiral swimming have
been reported. The aim of the present study is to clarify developmental mechanisms involved
in the mortality. SSH was used to generate sets of up- and down-regulated genes in M74-fry
at different stages of the syndrome. Amplified cDNAs were included in salmonid stress gene
array. Preliminary analysis of subtracted sequences and hybridisations between healthy and
diseased fry indicate differential expression patterns of genes belonging to several functional
categories including DNA and RNA metabolism, regulation of transcription, cell cycle and
growth, chaperone activity, myofibrils and hemoglobin complex.
PROTEIN ASSEMBLY OF PHOTOSYSTEM II AND ACCUMULATION OF
SUBCOMPLEXES IN THE ABSENCE OF LOW MOLECULAR WEIGHT
SUBUNITS PsbL AND PsbJ
Marjaana Suorsa1, Ralph E. Regel2, Virpi Paakkarinen1, Natalia Battchikova1, Reinhold G.
Herrmann2 and Eva-Mari Aro1
1
Department of Biology, Plant Physiology and Molecular Biology, FIN-20014 University of
Turku, Finland; 2Botanisches Institute der Ludwig-Maximilians Universität, Menzinger Str.
67, D-80638 München, Germany
Protein assembly and stability of photosystem II (PSII) (sub)complexes was studied in mature
leaves of four plastid mutants of tobacco (Nicotiana tabacum L.), each having one of the
psbEFLJ-operon genes inactivated. In the absence of psbL, no PSII core dimers or PSIILHCII supercomplexes were formed, and the assembly of CP43 to PSII core monomers was
extremely labile. The assembly of CP43 to PSII monomers was found necessary for the
assembly of PsbO on the lumenal side of PSII. The two other OEC proteins, PsbP and PsbQ,
were completely missing in ΔpsbL. In the absence of psbJ, both intact PSII core monomers
and PSII core dimers harboring the PsbO protein were formed whereas the LHCII antenna
remained detached from PSII dimers, as demonstrated by 77K fluorescence measurements
and by the lack of PSII-LHCII supercomplexes. The ΔpsbJ mutant was characterized by a
deficiency of PsbQ and a complete lack of PsbP. Thus both the PsbL and PsbJ subunits of
PSII are essential for proper assembly of the oxygen-evolving complex. The absence of psbE
and psbF resulted in a complete absence of all central PSII core and OEC proteins. On the
contrary, very young and vigorously expanding leaves of all psbEFLJ operon mutants
accumulated at least traces of D2 and the OEC proteins PsbO and PsbQ, implying a
developmental control in the expression of the PSII core and OEC proteins. Despite severe
problems in PSII assembly, the other thylakoid membrane complexes were present and
properly assembled in all psbEFLJ operon mutants.
POST-GENOMIC RESEARCH OF SYNECHOCYSTIS 6803 ctp GENE FAMILY
Tove Jansen
Department of Plant Physiology and Molecular Biology, University of Turku
The unicellular cyanobacterium Synechocystis 6803 is a Gram-negative prokaryote
performing oxygenic photosynthesis. Synechocystis 6803 has been widely used as a model
organism in photosynthesis research and it is believed to represent the chloroplasts ancestor.
The entire genome of Synechocystis 6803 was released already in 1996 and this launched the
post-genomic research of the organism.
The Synechocystis sp. PCC 6803 ctp gene family members ctpA (slr0008), ctpB (slr0257) and
ctpC (slr1751), encoding carboxyl-terminal endoproteases (Ctps), were studied at levels of
gene transcription and protein structure. Northern blot analysis revealed differential activation
and accumulation of the ctp transcripts upon induction of various environmental conditions,
including light, temperature, salinity and growth mode, supporting the view of distinct roles
of Ctps in Synechocystis sp. PCC 6803 cellular processes. Amino acid sequence comparison
of 16 ctp gene products showed that they fall into three distinct groups: the eukaryotic CtpA
like proteins, the prokaryotic CtpA like proteins and the prokaryotic CtpB/C like proteins.
Structural models of the Synechocystis sp. PCC 6803 Ctps, constructed based on the amino
acid sequence alignment and the crystal structure of the Scenedesmus obliquus D1 processing
protease, revealed that although the overall structure of the Synechocystis sp. PCC 6803 Ctps
is very similar, differences exist in the putative membrane contact regions and in the active
site environment.
PHOTOSYNTHETIC REDOX REGULATION OF ARABIDOPSIS NUCLEAR GENES
Mirva Piippo, Yagut Allahverdiyeva, Natalia Battchikova, Eva-Mari Aro
Dept. of Biol., Plant Phys. and Molec. Biol., Univ. of Turku, Turku, Finland
Various environmental conditions like temperature, light or nutrient stress are sensed in
chloroplasts as changes in the steady-state redox state of the photosynthetic electron transfer
components and/or of the coupled redox active components in the stroma. These redox signals
are thought to be conveyed to the nucleus in order to initiate the acclimation processes.
Redox-mediated cross-talk between the chloroplasts and the nucleus has earlier been
addressed particularly for the regulation of the lhcb genes. We have used Arabidopsis cDNA
microarrays to get a more comprehensive view on the regulation of nuclear genes by
chloroplast redox signals.
The redox state of chloroplasts was modulated by changing the quality and the quantity of
light, and by using wild type Arabidopsis leaves. Leaf samples were collected for gene
expression analysis as well as for various biochemical and biophysical measurements.
Within the first hours of illumination the expression of many genes encoding transcription
factors and stress-related proteins were altered, while changes in the expression of structural
genes were seen later. Several genes were specifically affected by various types of treatments
i.e. (i) by the reduction of the plastoquinone pool or (ii) by the preferential reduction of
electron acceptors on the reducing side of PSI or (iii) by the reduction of both the
plastoquinone pool and the electron acceptors on the reducing side of PSI. Detailed computer
analysis of the results obtained between different treatments is in progress to reveal the
patterns of gene regulation by chloroplasts redox signals.
NOVEL INSIGHTS INTO CYANOBACTERIAL MEMBRANES USING
PROTEOMICS APPROACH
Pengpeng Zhang
Department of Plant Physiology and Molecular Biology, University of Turku
Cyanobacteria are a group of photosynthetic prokaryotes performing oxygenic photosynthesis
and aerobic respiration in one cell compartment. They have three types of structurally and
functionally distinct membranes: the outer membrane, the plasma membrane and the
thylakoid membrane, where a number of macromolecular protein complexes are embedded.
The composition and dynamics of membrane complexes were studied in a unicellular
cyanobacterium Synechocystis sp. PCC 6803 by two-dimensional blue-native/SDS-PAGE
followed by MALDI-TOF. Approximately 20 distinct protein complexes could be isolated
from wild type cell membranes. They include the protein complexes involved in linear
photosynthetic electron flow and ATP synthesis (PSI, PSII, cytochrome b6f and ATP
synthase), four distinct complexes containing type I NAD(P)H dehydrogenase (NDH-1)
subunits, as well as several novel, unknown protein complexes. NDH-1 complexes are
involved both in the respiratory and photosynthetic electron transport chains, cyclic electron
flow, and inorganic carbon uptake. Their expression responded readily to changes in the CO2
concentration and other abiotic growth conditions, thus representing an extremely dynamic
group of membrane protein complexes. The dynamics of NDH-1 complexes was studied by
growing wild type and several mutant strains under various growth modes. The compositions
of NDH-1 L and M resemble each others, and they share most of subunits encoded by more
than 10 single copy genes (ndhAIGE, ndhB, ndhCJK, ndhH and ndhL) and two multigene
families (ndhD and ndhF). The relative amount of NDH-1 L and M is regulated by ambient
CO2 concentration. NDH-1 S complexes are completely different from NDH-1 L or M and
lack all the other NDH-1 subunits except NdhD3 and NdhF3. Their expression is induced by
limited CO2 condition and elevated pH. The existence of multiforms of NDH-1 is associated
with multifunction. NDH-1 L and M are probably mainly involved in respiratory electron
transfer and cyclic electron flow around PSI, while NDH-1 S, composed with NdhD3, NdhF3,
CupA and MBP70 is specifically associated with CO2 uptake.
GENOTYPIC ANALYSIS OF FLAVOBACTERIUM COLUMNARE ISOLATES IN
FINLAND
Lotta-Riina Suomalainen
Department of Biological and Environmental Science, P.O.Box 35, 40014 University of
Jyväskylä, Finland. E-mail: [email protected], tel. +358142604237
Flavobacterium columnare is the causative agent of columnaris disease in fish. First
outbreaks of columnaris disease occurred in Finland during 1990’s and during the past few
years the outbreaks have become more frequent and severe. High mortalities occur during
summer period especially among salmonid fingerlings, although recently some other reared
fish species have also been noticed to be susceptible to this disease. The outbreaks are usually
repetitive and consecutive antibiotic treatments are needed, which may lead to resistant strains
of the pathogen. The development of an effective vaccination has not been successful because
of the poor response of fish immune system to flavobacteria.
Four different genotypes of F. columnare are known in Asia and America. Genomic studies
are based on restriction analysis of 16S rDNA. European isolates of F. columnare have not
been genetically studied. Our aim is to survey Finnish isolates of F. columnare for differences
in their genotypes and virulence. This may give us a clue why F. columnare has made such a
massive invasion during last years. We assume that Finnish strains may be genetically
different and less virulent than Asian strains. Virulence may also differ between isolates from
different fish farms.
For this study we have received 29 F. columnare isolates collected by our research group,
National Veterinary and Food Research Institute and Dr. P. Rintamäki-Kinnunen. Isolates
have been collected from disease outbreaks from 15 different fish farms in Finland during 8
years. Methods such as 16S rDNA RFLP, LH-PCR, RISA and genomic RFLP were used to
distinguish different genotypes. So far RISA has been shown to be the best tool in genotypic
analysis; based on RISA we have identified 7 genotypes of F. columnare. AFLP and
sequencing of the full length 16S rDNA will be used later to get more detailed information on
the strains. Virulence testing will be done by infection studies on fish during next winter.
PARASITE AVOIDANCE BEHAVIOUR IN PREVENTING EYE FLUKE
INFECTIONS IN FISH
Anssi Karvonen, Otto Seppälä & E. Tellervo Valtonen
Department of Biological and Environmental Science, P.O. Box 35, FIN-40014, University of
Jyväskylä
This study investigated if rainbow trout, intermediate host for digenean parasite Diplostomum
spathaceum, could recognise the infection by the parasite and perform avoidance behaviour to
reduce the number of establishing parasites. We first performed controlled laboratory
infections and showed that fish acquired partial resistance to the parasite after first exposure,
which provided significant protection against subsequent infections. However, this resistance
was not sufficient to protect the fish against harmful effects of the infection when fish were
held in restricted cages in natural conditions implying that alternative defence mechanism is
needed. Results from behaviour experiments conducted in laboratory indicated that fish
responded to the parasite cercariae and moved away from the infection source, which suggests
that fish have mechanisms to avoid the infection. We suggest that fish benefit from
behavioural avoidance as it decreases the number of new infections and may also reduce
energetic costs otherwise needed for highly effective physiological resistance.
ALTERED BEHAVIOUR AND PREDATION VULNERABILITY OF FISH
INFECTED WITH TROPHICALLY TRANSMITTED PARASITES
Otto Seppälä, Anssi Karvonen & E. Tellervo Valtonen
Department of Biological and Environmental Science, P. O. Box 35
FIN-40014, University of Jyväskylä, Finland
Trophically transmitted parasites may increase their transmission efficiency by manipulating
behaviour of infected hosts so that their susceptibility to predation by target hosts is increased.
Diplostomum spathaceum (Trematoda) parasites reduce vision of their fish intermediate hosts
by locating in the eyes and inducing cataract formation, and thus have good potential to
manipulate fish behaviour. The aim of this study was to test whether D. spathaceum eye
flukes induce changes in fish swimming depth and escape behaviour, which could increase
fish vulnerability to predation by bird definitive hosts. Furthermore, the influence of parasites
on fish susceptibility to artificial above surface predators was studied. Here we show that D.
spathaceum eye flukes decrease fish escape behaviour, and increase fish vulnerability to
predation. We also examine the effect of the parasite developmental stage on fish predation
vulnerability, and discuss about possibility that altered host behaviour is an evolutionary
adaptation of the parasite to enhance transmission.
THE MULTIPLE APPLICATIONS OF THE IMMUNOCOMPETENCE-HANDICAP
HYPOTHESIS
Anssi Vainikka
Fish Biology & Fisheries Science, Department of Biological and Environmental Science
University of Jyväskylä
The immunocompetence-handicap hypothesis is probably one of the most debated and most
widely tested hypotheses in the field of immunoecology. It provides a mechanistic
explanation for the commonly observed negative correlation between parasite load and the
expression of secondary sexual characters (Hamilton & Zuk hypothesis): testosterone or an
analogous substance is needed to produce an ornament but it is immunosuppressive. Recent
studies have shown that also hormones like juvenile hormone in insects and 11ketotestosterone in cyprinids can have the effects that testosterone is known to have in many
vertebrates. On the other hand, testosterone seems not to be immunosuppressive in cyprinids.
In tench (Tinca tinca), testosterone was not found to suppress chemiluminescence response of
either blood or head kidney phagocytes or plasma lysozyme activity. In roach (Rutilus
rutilus), correlational data did not reveal negative relationships between testosterone and
several immune parameters (chemiluminescence and migration of head kidney phagocytes,
total and differential leukocyte counts and plasma IgM) while controlling the fish condition as
a partial correlate. Moreover, female cyprinids were observed to have at least equal
concentration of testosterone in comparison to males. Insects lack male-specific hormones
like testosterone, but an administration of juvenile hormone was found to increase pheromone
production in the grain beetle, Tenebrio molitor, while suppressing crucial immune functions
(encapsulation rate and phenoloxidase activity). The immunocompetence-handicap
mechanism seems to have a role in ensuring the honesty of secondary sexual signals in a wide
variety of taxa. However, the magnitude of ‘good genes’ effects seems to be dependent on the
intensity of sexual selection within a population and the evolutionary age of a species or its
co-evolutionary relationships with its parasites. Immunocompetence-handicap hypothesis in
condemned to fail if the Fisherian process maintains the puzzling ornamentation or if the
animal’s immunophysiology has adapted to high androgen levels.
THE EFFECTS OF BIOMANIPULATION – STUDY OF TEN EUTROPHIC LAKES
IN SOUTHERN FINLAND
Mikko Olin
Up to 2000 Finnish lakes are eutrophied and dominated by dense cyprinid fish stock.
Biomanipulation is nowadays very common method in restoring these lakes despite of the
difficulties and uncertainty involved. Firstly, it is difficult to estimate the cyprinid density or
how much fish has to be removed to improve water quality. Secondly, it is not known how
characteristics of Finnish lakes (e.g. short growing season, high turbidity) will affect to the
outcome. To answer these questions, Finnish Game and Fisheries Research Institute started an
extensive co-operative project called HOKA (1997-2002).
The HOKA-project includes ten study lakes or 14 lake basins. All the lakes have
suffered from blooms of blue-green algae (or Gonyostomum semen) and contained high
cyprinid biomass. The morphologic features and nutrient concentrations vary between the
lakes. The Cyprinid mass removal was conducted during years by motorised seining in the
autumn and fyke netting in the spring. Water quality studies included all important water
chemistry parameters, and phytoplankton and zooplankton surveys. Responses in fish
community were followed up by test fishing with NORDIC multimesh gillnets. The mass
removal target catch (200 kg/ha/3 years) was attained in five lakes/seven basins. Roach and
bream were the most common species. The catchability of white bream in seining appeared to
be low.
The gillnet catch per unit effort (CPUE) of large (>15 cm) cyprinids reduced in most
of the basins with sufficient mass removal catch (MRC). However, the CPUE of small (<10
cm) cyprinids increased in almost all basins. Especially roach and white bream effectively
compensated the removed biomass by rapid growth rate and massive reproduction. Bleak and
bream were the cyprinids that were negatively affected. The reproduction of perch and
pikeperch increased in many cases.
In the basins where the target catch was attained, the biomass of Cladocerans
increased clearly (>50 μg C/l) in five basins but reduced slightly in two. The biomass
decreased or retained in all basins where the target catch was not achieved. Biomanipulation
did not positively affect the mean length of Cladocerans. The percentage of Chydorus sp.,
indicators of eutrophication, decreased in all except one basin with sufficient mass removal
catch, and retained or increased in others. The total phytoplankton biomass reduced in six
basins with sufficient MRC and increased in one. In basins with low MRC, the total biomass
retained or increased. Particularly, the biomanipulation had negative effect on cyanobacteria:
the biomass decreased, the duration of blooms shortened and the biomass peak shifted
towards autumn. Total phosphorus (TP) concentration increased in most cases and was not
clearly related to mass removal catch. Total nitrogen (TN) concentration decreased or retained
in efficiently biomanipulated basins and increased in others. The turbidity was weakly
affected and none of the study lakes became notably clearer.
In conclusion, fishing was not efficient enough to prevent the recovery of cyprinids.
The target catch of 200 kg/ha/3 years is obviously too small in the most eutrophic lakes.
Despite the remaining of high cyprinid density, the cyanobacterial blooms clearly diminished.
The responses in zooplankton were probably too weak to be the only explanation. Another
possible reason is changes in nutrient cycling, as chl:TP ratios decreased in most cases.
Decrease of TN and chl:TN ratio can indicate an increase of macrophytes. However, direct
evidence of macrophyte recovering was available only from lake Tuusulanjärvi. The
enhanced growth rate of heavily harvested cyprinid stock may induce a shift where cyprinids
start to act more like “nutrient-sink” instead of “nutrient-recycler”.
SPAWNING HABITAT REQUIREMENTS OF RIVERINE EUROPEAN GRAYLING
THYMALLUS THYMALLUS L.
Mari Nykänen1 and Ari Huusko2
Department of Biological and Environmental Science, University of Jyväskylä
Finnish Game and Fisheries Research Institute, Kainuu Fisheries Research and Aquaculture
1
2
European grayling is threatened in many parts of its distribution area by damming and
channelization of rivers. One of the main reasons for population declines is considered to be
lack of suitable spawning habitat. In order to conserve and enhance grayling habitats and thus
populations, we need to know what kind of habitats the fish need as spawning sites. It is often
best to use site-specific habitat criteria as a basis of river enhancements. However, criteria
development is expensive and labour intensive. Moreover, if a river is not close to its natural
state, there may be only a few fish to monitor, and the on site criteria may not reliably
represent the conditions that are truly preferred by the fish. Therefore, generalised suitability
criteria applicable to a wide range of conditions are also needed. The aims of the present study
were 1) to determine the spawning habitat preferences of grayling in the River Kuusinkijoki, a
medium-sized boreal river, and 2) to develop generalised suitability criteria for spawning sites
by combining the present information with data available in literature. In the River
Kuusinkijoki, spawning sites of grayling were searched for by kick sampling, and the
characteristics of the egg burial sites were then compared to characteristics of the available
habitat to determine habitat preference curves for depth, mean velocity, bottom velocity, and
vegetation cover. Data in literature were converted into corresponding curve format and the
curves for each habitat characteristic were then averaged. The generalised optimal ranges for
dominant substratum size, water depth, mean velocity, bottom velocity, and percent instream
vegetation cover at egg burial sites of grayling were 16-32 mm, 30-40 cm, 50-60 cm s-1, 40-50
cm s-1, and <10 %, respectively. Whereas the criteria for velocity and substratum appear to be
relatively fixed, the role of depth in the spawning site selection of grayling requires further
investigation.
TEMPORAL CORRELATIONS IN THE LEAF CHEMISTRY OF MOUNTAIN
BIRCH: ARE THE SAME TREES ALWAYS RESISTANT TO HERBIVORES?
Riipi, M., Haukioja, E., Lempa, K., Ossipov, V., Ossipova, S. & Pihlaja, K.
The quality of tree leaves as food for herbivores changes rapidly especially during the spring
and early summer. However, whether the quality of an individual tree in relation to other trees
in the population changes during the growing season and between years is less clear. We
studied the seasonal and annual stability of chemical and physical traits affecting leaf quality
for herbivores. Rankings of trees in terms of the contents of two major groups of phenolics in
their leaves, hydrolyzable tannins and proanthocyanidins (condensed tannins), were very
stable from the early spring to the end of the growing season. There were also strong positive
within-season correlations in the levels of some other groups of phenolics in the leaves
(kaempferol glycosides, myricetin glycosides and p-coumaroylquinic acid derivatives). The
contents of individual sugars and the sum content of protein-bound amino acids showed
patterns of seasonal consistency in mature leaves, but not in young developing leaves. The
seasonal correlations in leaf water content and toughness were also strongest in mature leaves.
The correlations between two years at corresponding times of the growing season were
strongly positive for the major groups of phenolics throughout the season, but were more
variable for the contents of proteins and some sugars. Leaf toughness and water content
showed strong positive correlations in mature leaves. Despite the consistency of tree ranking
in terms of leaf phenolics, the relative resistance status of trees may, however, change during
a growing season because there was a negative correlation between the content of
hydrolyzable tannins (early-season resistance compounds) in leaves early in the season and
the content of proanthocyanidins (late-season resistance compounds) late in the season, and
vice versa. Thus, assuming that phenolics affect herbivore preference and performance,
different plants may suffer damage at different times of the growing season, and the overall
variation between trees in the fitness consequences may be low. In addition, the adaptation of
herbivorous insects to mountain birch foliage in general, as well as to specific tree
individuals, may be constrained by variation in the relative resistance status of the trees.
SHADING INDUCED VARIABILITY IN MOUNTAIN BIRCH LEAF CHEMISTRY –
EFFECTS ON PERFORMANCE AND FAECAL COMPOSITION OF SAWFLIES
Lauri Kapari
I shaded large branches of mountain birch and studied growth and consumption of larvae of
six species of sawflies on shaded and non-shaded leaves of 14 birch trees. I also analysed both
leaf and faecal phenols of larvae in each tree. Branch shading resulted on average better
quality foliage for the sawflies, and all the species performed best on same types of leaves,
characterized by low concentrations of phenolic compounds and high concentrations of water
and amino acids. The effects of leaf quality on larval consumption varied between early and
late season species: the former consumed less on low quality foliage, while the latter showed
different degrees of compensatory (increased) consumption to low leaf quality. A new finding
was that many phenolic compounds in insect faeces were as good or better predictors of
foliage quality than foliar phenols. The concentration of N-rich dihydroxyphenylurea in
faeces had only few correlations with larval traits. Some new, non-foliar flavonoid
compounds were found in faeces, but their occurrence did not have any clear effects on larval
performance.
HETEROLOGOUS HEMOGLOBIN (vhb)-GENE EFFECTS ON PATHOGEN
RESISTANCE AND MYCORRHIZAL INTERACTION IN HYBRID ASPEN
(POPULUS TREMULA X TREMULOIDES)
Soile Jokipii
University of Oulu
Hybrid aspen is an artificial cross between European (Populus tremula) and American aspen
(P. tremuloides) that has superior growth characteristics compared to the both parent species.
Special industrial interest in aspen and hybrid aspen as a source of mechanical fibre and in
pulp processing has rosen quite recently. Populus species including hybrid aspen have also
proved to be competent for micropropagation and genetic transformation indicating the
possibilities for molecular breeding work.
Non-symbiotic hemoglobins and their function in plants are not well known today. It is
hypothesized that their role might include facilitation of oxygen diffusion, oxygen sensing or
terminal oxidase activity. Our previous studies on expression of heterologous Vitreoscilla
hemoglobin (VHb) in hybrid aspen indicated connection of energy metabolism in VHb action.
We also observed spontaneous infections by a fungal pathogen in greenhouse pointing to
higher resistance in transgenic VHb-expressing lines than in non-transgenic control lines. In
the present work we intend I) to study the effects of pathogen infections on VHb expressing
and control lines; II) to study the effects of vhb gene on mycorrhizal symbiosis and; III) to
study the potential resistance mechanisms of hemoglobin action with the help of heterologous
vhb gene and endogenous hemoglobin genes to be cloned and characterized.
IMPORTANCE OF CARDIAC POTASSIUM CHANNELS IN TEMPERATURE
ACCLIMATION OF RAINBOW TROUT HEART
Minna Hassinen & Matti Vornanen
University of Joensuu, Department of Biology, P.O.Box 111, FIN-80101 Joensuu, Finland
Temperature is an important factor affecting both distribution and survival of animals. While
endotherms maintain constant body temperature with high expenditure of energy, ectotherms
selectively regulate key body functions in temperature-dependent manner and thus avoid
wastage of energy. Rainbow trout (Oncorhynchus mykiss) is a relatively eurythermic species
which remains active in cold waters. Active life style means that tissue cells have a constant
need for oxygen and nutrients, and thus the cardiovascular function must be maintained at
high level even in the cold. Indeed, temperature adaptation of cardiac function in rainbow
trout seems to involve some kind of acclimation program, which includes several plastic
components, e.g. changes in myofilament function, sarcolemmal K+ currents and Ca-pump of
the sarcoplasmic reticulum.
Potassium channels are important in maintaining the resting membrane potential of
cardiac myocytes, determining the refractoriness of the cardiac contraction and regulation of
the action potential duration. Two major K+ currents of the trout heart are the background
inward rectifier (IK1) and the rapid component of the delayed inward rectifier (IKr). IK1 helps to
maintain the resting potential near the equilibrium potential for K+ and IKr is important in the
repolarization of the cardiac action potential. In cold acclimated trout, these two currents are
modified by opposite manner: IK1 is depressed and IKr is up-regulated. As a final outcome of
these changes the action potential duration becomes shorter in the cold-acclimated fish heart
which enables higher heart rate in the cold. Electrophysiological measurements suggest that
the amplitudes of the currents are changed but the kinetics of the currents remain constant.
Thus, the changes in current size are supposed to be dependent on the number of the channels
in the cell membrane. The molecular basis of the IKr is the ERG channel, a voltage dependent
channel of the plasma membrane. IK1 may involve several types of inwardly rectifying
channels: at least Kir2.1 and Kir2.2, but prehaps also of Kir2.3. These three Kir-channels have
different K+ conductances and the composition of Kirs has been shown to change during
cardiac development in many species.
The aim of our studies is to clarify how these potassium channels contribute to
temperature adaptation of rainbow trout and how their expression is regulated in changing
environmental conditions. The first aim is to clone the genes coding these channels. The
genes of rainbow trout ERG and Kir2.1 channels have alredy been sequenced almost
completely. The cloning was done by RT-PCR using degenerative primers and cDNA from
the rainbow trout heart as template. PCR-products were cloned into the pGEM-T Easy vector
and transformed into Escherichia coli DH5α-cell line. One clone was selected by PCR and
grown in LB-medium. The plasmids were isolated by Wizard midiprep plasmid purification
kit and sequenced by ABI Prism DNA sequencer using the ABI Prism BigDye terminator
cycle sequencing ready reaction kit.
Next, the expression of these potassium channel genes in different environmental conditions
will be studied by northern hybridization and in situ hybridization. It will be interesting to see
whether the different Kir-channels have different roles in environmental temperature changes.
The regulation of these genes is to be studied by cloning the promoters of the genes and
characterizing their transcription elements by computer database studies. The putative
transcription factor binding sites will be studied by EMSA. CDNA microarray analysis will
also be done to compare the gene expression levels of cold- and warm-acclimated rainbow
trout's atrium and ventricle, which may give new viewpoins to the research.
DEVELOPING YEAST CELL-BASED ASSAYS FOR MEASURING ENDOCRINE
DISRUPTERS IN ENVIRONMENT
Piia Leskinen
University of Turku, Department of Biotechnology
Tykistökatu 6, 6th floor, FIN-20520 Turku, +358-2-3338059, [email protected]
Endocrine disrupters can be defined as exogenous substances that cause adverse health effects
in an intact organism, or its progeny, subsequent to changes in endocrine function. Such
chemicals do not necessarily exhibit structural similarities and thus they cannot be measured
by chemical methods. In vitro methods using living cells are needed to identify chemicals that
have endocrine disrupting properties and to assess the potential of chemical mixtures found in
environment to affect the hormonal systems of organisms.
In this project new yeast based tests are developed using firefly luciferase (luc) as a reporter
of ligand induced activity of steroid receptors. The yeast tests are simple and inexpensive to
use compared to tests based on mammalian cells. They are suitable for screening of chemicals
or environmental samples for their capability of activating steroid receptors. However, some
chemicals can affect the hormonal system through other pathways than through direct
interaction with steroid receptors and thus in-vivo tests will be needed to confirm the results of
yeast tests. So far yeast cells for measuring androgenic, estrogenic and glucocorticoid activity
have been constructed. The tests have been successfully applied for measuring androgenic and
estrogenic activity of certain chemicals and of influents and effluents of waste water treatment
plants.
THE EXPRESSION OF STEROIDOGENIC ENZYMES IN THE
DEVELOPING OVARY OF CONTROL AND IN UTERO TCDDEXPOSED SPRAQUE-DAWLEY RAT
1
Sari Myllymäki, 1Tapio Haavisto, 2Matti Viluksela, 3Jorma Toppari, 1Jorma Paranko
1
Laboratory of Animal Physiology, Department of Biology, University of Turku,
Finland
2
Laboratory of Toxicology, National Public Health Institute, Kuopio, Finland
3
Department of Physiology, University of Turku, Finland
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxicant with the ability to disrupt
endocrine and reproductive systems. Half-life in the human body is extremely long, ca. 7
years. Despite numerous studies to characterize the general toxicity of TCDD in reproductive
and other tissues, little is known about the mechanisms by which TCDD exerts its effect at the
cellular and molecular level. It has been reported that TCDD may inhibit steroidogenesis at
specific steps like the mobilization of cholesterol from the cytoplasm to the inner
mitochondrial membrane and the activity of steroidogenic P450 enzymes. The present
experiments were designed to test the effects of in utero and lactational TCDD exposure on
ovarian steroidogenesis by analysing mRNA levels of several steroidogenic enzymes.
1 µg/kg TCDD was given orally to Spraque-Dawley rats on gestational day 13. On the
postnatal day 1, litters were adjusted to a group of four females and four males. Pups were
killed on the postnatal days 10, 12, 14, or 16 and ovaries were snap frozen for RT-PCR.
Plasma samples were collected for estradiol, testosterone, progesterone, LH, and FSH
measurements. After ether extraction, estradiol, testosterone, and progesterone levels were
measured by dissociation-enhanced lanthanide fluoroimmunoassay; LH and FSH were
measured by an immunofluorometric assay (DELFIA, Wallac). The expression of StAR
(Steroidogenic
acute
regulatory
protein),
3beta-HSD1
(3-beta-hydroxysteroid
dehydrogenase/delta-5-delta-4 isomerase type I), P450scc (Cyp11a, cholesterol side-chain
cleavage), P450-17alfa (Cyp17, 17-alfa-hydroxylase/17,20-lyase) and P450arom (Cyp19,
Aromatase) was assessed by using two-step real-time RT-PCR (QuantiTect SYBR Green
PCR Kit, Qiagen). Total RNA was isolated from ovary using RNA isolation kit (Qiagen) and
Qia Shredder column for tissue lysate homogenization. Two micrograms of total RNA and
Oligo(dT)15 primer were incubated with 30 units of AMV reverse transcriptase at 42 C for 1
h. RT-PCR for ribosomal S26 was used as a control for the amount of RNA used.
The present study shows that the female steroidogenic system is sensitive to TCDD exposure.
Interference could be demonstrated as inhibition of estradiol synthesis, stimulation of FSH
and LH production, and decreased mRNA levels of steroidogenic enzymes, StAR, P450scc,
and P450arom.