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Kristiina A. M. Vuori and Mikko Nikinmaa
M74 Syndrome in Baltic Salmon and the
Possible Role of Oxidative Stresses in Its
Development: Present Knowledge and
Perspectives for Future Studies
Baltic salmon suffer from maternally transmitted yolk-sac
fry mortality syndrome—M74. The inc idence of M74
varies considerably on a year to year basis. In the
1990s the mortalities were 50–80% but in 2003–2005,
below 10%. Befo re death, M74-affected fry have several
typical symptoms. M74-eggs are characterized by low
thiamine and carotenoid content, and affected fry show
signs of oxidative stress. Although M74 is associated with
thiamine deficiency and the symptoms of the fry can be
alleviated with thiamine, the underlying causes of the
syndrome have remained a mystery. We have studied the
symptoms of M74 at the molecular level by investigatin g
the global gene expression patterns using cDNA micro-
array and have quantified the changes in transcriptional
regulation in M74-affected and healthy yolk-sac fry. Our
and previous resu lts sugges t that M74 in Baltic salmon
yolk-sac fry results from oxidative stresses disturbing
several different developmental mole cular pathways.
Because the M74 syndrome is of maternal origin, fact ors
in the Baltic Sea during salmon feeding and migration,
i.e., the chemical composition of food, may be decisive in
the development of M74. The possible mechanisms by
which oxidative stresses may develop in adult salmon are
discussed in the review.
WHAT IS M74: A DESCRIPTION OF ITS OCCURRENCE
AND MAJOR SYMPTOMS
The yolk-sac fry mortality syndrome of Baltic salmon (Salmo
salar) was first described in 1974 in Swedish compensatory
hatcheries. Thereafter the syndrome has been designated as the
M74 syndrome (M ¼ miljo
¨
betingad, environmentally induced;
[1]). M74 has been observed in the yolk-sac fry of feral Baltic
salmon in several rivers of Sweden and Finland, but not in
Latvian or Polish rivers (1–5). A similar type of fry mortality
syndrome (early mortality syndrom e [EMS]) is found in
salmonids of the North American Great Lakes and New York
Finger Lakes (6). The incidence of the M74 syndrome is
variable. Analysis of long-term mortality records from two
Swedish Baltic salmon hatchery stocks in the years 1928–1998
indicated low or no occurrence of M74 before 1974 (7). In the
1990s, 25–80% of Baltic salmon females, which ascended rivers
to spawn, produced yolk-sac fry suffering from the syndrome
(2, 3, 8). In the years 2003–2005, only a few percent of the
salmon fry suffered from the syndrome, but a current analysis
monitoring the level of M74 in 2006 suggests there is a higher
incidence than in previous years (9). Whereas most of the
observations of the syndrome have been made in offspring that
originate from artificially fertilized eggs intended for stocking,
there are stron g indications from electrofishing and parr
abundance estimations from Swedish rivers that M74 has also
affected naturally spawning populations during the periods of
high occurrence (3, 10).
M74 is maternally transmitted and affects the yolk-sac stage
of the fry. Within an affected family group the fry mortality is
often 100% although groups of partial mortality have also been
described (1, 2, 8). M74-affected fry have several typical
neurological, cardiovascular, morphological, and other symp-
toms: They show a disturbed swimming pattern, impaired
coordination, a lack of phototaxis, and a decreased heart rate.
The absorption of yolk is slowed down. The M74-fry are also
characterized by a small, pale spleen and show blood
congestion, a reduced number of circulating erythrocytes,
abnormal hemorrhages/blood coagulation, exopthalmia, glyco-
gen depletion, and an increased number of necrotic cells in the
brain. Dying yolk-sac fry are lethargic and have convulsions
and bradycardia (1, 11, 12). M74-producing brood fish may
show wiggling behavior that is most likely caused by alterations
in dopaminergic and serotonergic activity in the brain (13).
M74 is associated with a low thiamine content in the brood
fish and eggs (2, 14, 15). The symptoms of M74 can be treated
with thiamine and induced with thiamine antagonists (16–18).
Consequently, it has been suggested that a decrease in the
thiamine content of food or an increase in its thiaminase activity
could be the cause of the syndrome (19, 20). However, there are
presently no conclusive data showing that the thiamine content,
or thiaminase activity of the food of salmon, would have
changed during the recent past (19, 20). Alternatively, the
accumulation of xenobiotics has also been suggested as the
cause of the syndrome (2, 21). However, the published data
about the correlation of organochlorine concentrations in the
muscle of female salmon and fry mortality is contradictory (21,
22), and direct connection between organochlorine accumula-
tion in broodstock and following effects on females an d
developing fry has not yet been shown.
OXIDATIVE STRESSES MAY BE ASSOCIATED WITH
THE M74 SYNDROME
In addition to reduced thiamine levels, M74 is usually
associated with reduced levels of antioxidants such as astax-
anthin, a-tocopherol, and ubiquinone (2, 23, 24). Furthermore,
the cellular reduced/oxidized glutathione ratio of the M74-fry
(GSH/GSSG) is altered in favor of the oxidized form (25), and
the activities of redox enzymes in the liver (glutathione
peroxidase, glutathione reductase, and glutathione-S-transfer-
ase) are increased (25, 26). M74-eggs also have more oxidized
fatty acids than healthy ones (27, 28). Histopathological
features of M74-fry, such as hepatocellular lipid accumulation,
dilatation of hepatocyte endoplasmic reticulum, and degenera-
tion of skeletal muscle, are also characteristic of long-term
oxidative stress (12). These findings suggest that disturbances in
the redox state of the fish are associated with M74. Further-
more, thiamine deficiency itself has repeatedly been shown to be
caused by oxidative stresses (e.g. 29, 30), and thiamine addition
reduces reactive oxygen species (ROS)–induced damage by
168 Ambio Vol. 36, No. 2–3, April 2007Ó Royal Swedish Academy of Sciences 2007
http://www.ambio.kva.se
scavenging them (31). Taken together these data suggest that
thiamine deficiency can be an indicator of prior oxidative stress.
POSSIBLE REASONS BEHIND THE M74 SYNDROME
IN THE BALTIC SEA
As stated above, M74 is transferred maternally and eggs of
M74-producing females have lower levels of thiamine and
carotenoids than eggs of females producing healthy offspring
(15, 24, 27, 28). Thus, factors in the Baltic Sea, which adult
salmon experience during their feeding migration, are decisive
for the development of M74. Karlsson et al. (19), Hansson et al.
(7), and Pickova et al. (28) have suggested that changes in the
properties of the food web affecting the chemical composition
of salmon prey may be one of the key factors that cause M74.
Changes in the food web could be associated with recent
eutrophication and decreasing salinity of the Baltic Sea. Both
changes in salinity (32) and eutrophication (33) affect the
dynamics and composition of phytoplankton communities and
their cellular levels of antioxidants. In addition, a general shift
from a more neritic toward a more limnic zooplankton
community has occurred because of a decreased inflow of
saline water (34, 35), and it is known that the zooplankton
species composition in the Baltic Sea varies according to salinity
gradients (36). It is suggested that neritic zooplankton species
offer a better source of astaxantin than limnic species (37). The
differences in the phytoplankton and zooplankton between the
Baltic Sea and Norwegian Sea also appear compatible with the
occurrence of M74 in Baltic but not in oceanic salmon (38).
The changes in phytoplankton and zooplankton may reflect
changes in growth and quality of prey species eaten by Baltic
salmon. More specifically, since carotenoids and thiamine in the
salmon diet originate from phytoplankton, any decrease in the
production or transformation of these compounds because of
changes in the composition of plankton communities could
affect the supply of these nutrients that are available to salmon
via plankton and prey fish (39). However, in the case of
thiamine, the concentrations in the Baltic Sea plankton do not
differ from other coastal areas (39) and concentration in salmon
prey sprat and herring have been assessed as adequate for the
salmon diet (40). Therefore, metabolic proc esses such as
enhanced redox enzyme activities and increased biotransforma-
tion may have important roles in the development of M74, as
they may deplete the antioxidant pool of brood fishes (41, 42)
and ultimately deplete thiamine levels, as discussed above.
In order to evaluate the role of different abiotic and biotic
factors in the Baltic Sea in causing the syndrome, it is important
that correlations between the incidence of M74 and its potential
causative factors (e.g. variation in plankton species composi-
tion) in the feeding areas of the salmon populations are
established. To our knowledge, such information is not
available presently in the published literature. Another factor
that needs to be considered is the general trend of decreased
salinity in the Baltic Sea during the latter part of the1900s, with
intermittent pronounced salinity increases associated with
irregularly occurring ‘‘saltwater pulses’’ (34, 43). If a change
in the salinity and its associated changes in the food web played
a role in the development of the M74 syndrome, then one could
expect the variable incidence of the syndrome to follow salinity
shifts.
The association of organochlorine toxicants with the
development and incidence of M74 is still unresolved. Asplund
et al. (22) observed no differences in the concentrations of
dichlorodi phenyltrichl oroethan e (DD T) an d re lated com-
pounds, polychlorinated biphenyls (PCBs), polybrominated
diphenyl ethers, hexachlorobentzene, or methoxylated bromi-
nated diphenyl ethers, between healthy salmon and salmon that
produced offspring with M74 using samples that were collected
from the river Dala
¨
lven in 1995. In contrast, Vuorinen et al. (21)
reported a correlation between polychlorinated dibentzofurans,
coplanar PCBs, 2,3
0
,4,4
0
,5-pentachloro diphenyl ether, oxy-
chlordane, cischlordane, hexachlorobentzene, dichlorodiphenyl-
dichloroethylene, and M74 mortality using samples that were
collected from the river Simojoki in 1988–1992. It has been
reported that the total concentrations of PCBs, DDT,
polychlorinated dibenzo-p-dioxins and -furans (PCDF) in the
Baltic Sea vertebrates have decreased from the 1970s (44–47).
However, it is suggested that concentrations of certain PCDFs
and coplanar PCBs still remain high in salmon and its diet
because of changes in prey stock sizes in favor of sprat and
decreased sprat and herring growth (40). In addition, among the
organic xenobiotics, polybrominated biphenyls, used in flame
retardants, are still used, and there may be increasing
concentrations of these toxicants in the Baltic Sea (48). At
present, however, neither the concentrations of these com-
pounds in the feeding grounds of salmon in the Baltic nor
information about the effects of these compounds in fish at
environmentally relevant concentrations are available.
MOLECULAR STUDIES ON YOLK -SAC FRY: NEW
INFORMATION ABOUT THE DEVELOPMENT OF
SYMPTOMS INCLUDING THE ROLE OF OXIDATIVE
STRESSES
Because the symptoms of M74 are observed in developing fry,
most studies have concentrated on the disturbances observed in
fry, although, as pointed out above, the internal environment of
the adult and the conditions experienced by the adult are
instrumental in the development of the syndrome. As indicated
by the many symptoms observed in M74-fry, it is clear that
many sets of developmental pathways are affected. We have
investigated the global gene expression patterns during devel-
opment by cDNA microarray (49) and examined the changes in
transcriptional regulation (50) between M74-affected and
healthy yolk-sac fry.
On the basis of microarray and earlier mortality data (2, 26),
the fry suffering from M74 syndrome can be divided into three
groups with either early onset, intermediate onset, or late onset
mortality. Each M74-subgroup has a unique gene expression
pattern at the preclinical and clinical stage, which precedes
terminal responses characterized by a transcription profile that
is explaine d by a n inh ibitio n of the cell cyc le and cell
proliferation, and consequent cell death. If the disturbance
occurs early, i.e. death occurs during the first third of the yolk-
sac stage, virtually all of the responses are compatible with
immediate stress, which rapidly leads to the common terminal
responses. If the death occurs during the second third of the
yolk sac stage (intermediate group), the terminal stage is
preceded by a clear disturbance in globin gene expression, which
will lead to internal hypoxia, when the fry grow and shift from
the predominantly skin-breathing to the gill-breathing stage of
development. In the absence of compensations for reduced
oxygen delivery, the group will then proceed to the terminal
responses. The slowest group to develop M74 (late group)
appears to compensate for reduced oxygen delivery by slowing
down metabolism, and hence some fry can escape death (Fig. 1).
Symptoms of M74-affected fry include a group of symptoms
associated w ith the development and maintenance of the
vasculature and circulation (impaired vascular development, a
reduced number of circulating erythrocytes, and abnormal
hemorrhages/blood coagulation [1, 11, 12]). Similar defects are
observed in mammalian embryos if the function of transcription
factor hypoxia inducible factor a (HIF-1a), its dimerization
partner aryl hydrocarbon nuclear translocator, or target gene
Ambio Vol. 36, No. 2–3, April 2007 169Ó Royal Swedish Academy of Sciences 2007
http://www.ambio.kva.se
vascular endothelial growth factor (VEGF), is disturbed (51–
53). In addition, necrotic cells are observed in the brain of M74-
fry (12), and various neurological disorders have been reported
(1, 11, 12). Similarly, in mammalian embryos, neurological
development is disturbed and full mortality is observed if HIF-
1a is nonfunctional (51). Nikinmaa et al. (54) have shown that
salmonid HIF-1a is sensitive to redox disturbances such as
oxidative stresses. Furthermore, we established that the
intermediate type of M74 mortality is associated with reduced
DNA-binding of HIF-1a, reduced production of the VEGF
protein, and decreased capillary density (Fig. 2). These results,
together with the downregulation of the adult-type hemoglobin
gene transcription, indicated that M74-fry experienced internal
hypoxia at the time that the fry shifted from skin- to gill-
breathing, at which time they probably also changed from the
production of larval- to adult-type hemoglobins.
Because organochlorine toxicants, implicated in the devel-
opment of M74 (21), are associated with the induction of the
aryl hydrocarbon receptor (AhR)-dependent gene expression
pathway, we have also studied its expression in healthy and
M74-affected fry. The AhR target gene CYP1A protein, often
determined in ecotoxicological studies, is expressed at high
levels in the Baltic salmon as compared to hatchery-reared fry,
indicating that the Baltic Sea is a highly contaminated
environm ent (50). However, l ess AhR DNA-binding and
CYP1A protein were seen in the intermediate group M74-fry
than in healthy fry, suggesting that the induction of an AhR-
dependent pathway does not cause the M74-fry mortality
(Fig. 3). These results are consistent with the results of
Lundsdtro
¨
m et al. (26), which indicated decreased ethoxyresor-
ufin-O-deethylase activity in M74-fry as compared to healthy
fry. AhR-dependent gene expression may be involved in cell
cycle regulation and developmental processes (for reviews see 55
and 56), which are disturbed in M74-fry. For example, AhR has
been shown to regulate vascular and neuronal development (e.g.
57, 58). In accordance with the many roles of AhR in
development, AhR expression was observed in the spinal cord
and brain, liver, muscle, gut epithelium, and head cartilage of
healthy wild and hatchery-reared Baltic salmon yolk-sac fry (K.
A. M. Vuori, J. Kallio and M. Nikinmaa, unpubl. data). Thus,
it is likely that the decreased function of AhR-dependent
developmental pathways could be partly responsible for the
symptoms associated with the M74 syndrome. However, the
factors causing reduced AhR function in M74-fry are unknown
at present.
OXIDATIVE STRESS IN SALMON DURING FEEDING
MIGRATION
If oxidative stresses play a role in the development of the M74
syndrome, then differences in the metabolism associated with
oxidative stresses should be evident in actively feeding adult
females before spawning migrations because the syndrome is of
maternal origin. We are not aware of published data on
oxidative stress parameters and their variability in feeding Baltic
salmon from different populations. In this regard, e.g. the
enzymes involved in handling ROS, the products of oxidation in
organisms, are important. The key enzymes for the detoxifica-
tion of ROS in all organisms are superoxidase dismutase,
glutathione peroxidase, peroxidase, and catalase. This enzyme
battery is supplemented by systems providing reducing equiva-
lents needed for detoxifying activity (e.g. glutathione reductase,
glutathione-s-transferase; glucose 6-phosphate dehydrogenase).
Figure 2. The vascular defect s
observed in M74-affected fry are
associate d wi th red uced DNA-
binding of transcription factor
HIF-1a and subsequent downregu-
lation of VEGF, involved in angio-
genesis.
Figure 3. M74-affected fry show
reduced DNA-binding of transcrip-
tion factor AhR followed by subse-
quent downre gulation of CYP1A
and a decrease of EROD activity.
*from Lundstro
¨
m et al. (26).
Figure 1. Proposed molecular
events leading to mortality in
M74-fry dying at different periods
of yolk-sac fry development.
170 Ambio Vol. 36, No. 2–3, April 2007Ó Royal Swedish Academy of Sciences 2007
http://www.ambio.kva.se
In addition, low molecular weight radical scavangers (e.g.
glutath ione) and antioxidants (e.g. b- caro tene, vitamin E)
contribute to protection against ROS (reviewed in e.g. 41, 42).
Our preliminary analyses of a sample set of feeding Baltic
salmon indicate a marked (3–20-fold) size-independent and size-
dependent variation in many of the abovementioned redox
parameters (M. Kanerva, M. Nikinmaa and K.A.M. Vuori,
unpubl. data). Further studies about individual differences in the
oxidative stress status of feeding salmon are needed, using fish
from several feeding areas and estimating their genetic back-
ground, before firm conclusions can be made of the possible
association of M74 and oxidative stresses in salmon during the
feeding migration.
HOW VARIATION BETWEEN INDIVIDUALS AND
POPULATIONS MAY AFFECT RESPONSES
One notable feature in the occurrence of M74 is that there is
large variation in the susceptibility of the offspring of different
individuals to the syndrome (2, 49). However, at present, the
role of individual variability, or possible differences between
populations, in the biochemical pathways involved in, e.g.,
handling oxidative stresses has not been studied, although
differences in the prevalence of M74 between Finnish/Swedish
and Latvian/Polish populations have been described.
CONCLUSIONS
At the moment, several lines of research support the hypothesis
that oxidative stresses cause the symptoms that are part of the
M74 syndrome, and the effects of oxidative stress can be
transmitted from the parent to the egg. Hitherto, factors
causing oxidative stresses for Baltic salmon populations have
remained elusive, but may be related to changes in the food
web, possibly to alterations in plankton communities as a result
of eutrophication, and/or salinity changes of the Baltic Sea.
There is p rono unced vari atio n in th e occur renc e of the
syndrome between individuals and populations. However, the
basis of this variation is completely unknown, making it
especially important for future studies to examine the variability
in the responses of feeding salmon to oxidative stresses.
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Kristiina A. M. Vuori is a Ph.D. student at the University of
Turku, Finland. She holds a M.Sc. in Animal Physiology from
University of Turku. Her research interests are the molecular
background of M74 and the general application of molecular
and physiolo gical me thod olo gy to aquati c envi ronm enta l
research. Her address: Center of Excellence in Evolutionary
Genetics and Physiology, Department of Biology, University of
Turku, FI-20014 Turku, Finland.
E-mail: kristiina.vuori@utu.fi
Mikko Nikinmaa is a professor of Zoology in the Department of
Biology, University of Turku. He is the coeditor-in-chief of
Aquatic Toxicology, and director of the Center of Excellence in
Evolutionary Genetics and Physiology. His research interests
focus on environmental regulation of gene expression,
especially of oxygen-dependent phenomena in fishes. His
address: Center of Excellence in Evolutionary Genetics and
Physiology, Department of Biology, University of Turku, FI-
20014 Turku, Finland.
E-mail: miknik@utu.fi
172 Ambio Vol. 36, No. 2–3, April 2007Ó Royal Swedish Academy of Sciences 2007
http://www.ambio.kva.se
