Changes in lipid and fatty acid content
The average lipid content in the muscle of the salmon at the beginning of the spawning run from the Baltic Proper in the spring tended to be lower than in the muscle of salmon that had been caught from the same area during their feeding migration in the late autumn (8.7%; Keinänen et al., unpubl.). The lipid content of prey fish decreases by the spring [1, 7, 67, 80], and the feeding activity of salmon decreases at the same time [13]. The salmon from the present study that were caught in the spring had probably already used part of their lipid stores acquired during high autumn feeding activity.
Although the muscular lipid content did not differ between the sampling locations in the sea, it halved by spawning time. This decrease was in the same range as reported for salmonine species during the pre-spawning period in general [6]. In 1–3 sea-year salmon, even a decrease of 60% in muscle lipid content was found from the start of the spawning run in the Baltic Proper till the spawning in the River Simojoki [18]. In the present study’s spawning salmon, the muscle lipid content (3.65 ± 0.78%) was much lower than in those salmon sampled at spawning in 1991 and 1992 (means of 7.58 and 9.45% respectively) [81], i.e., in the early years of the highest incidence of M74 [4, 11].
The activity of hepatic G6PDH, which is known to decrease with increasing dietary lipids [82,83,84], did not differ significantly between salmon caught at all locations. However, in those spawning salmon that had been feeding in the Baltic Proper the G6PDH activity was approximately 50% and significantly lower than in salmon captured from the Baltic Proper, probably due to the mobilisation of FAs from body lipids for metabolism, the development of oocytes, and various kinds of physical activity. Thus, lipids from body stores probably affected hepatic G6PDH activity similarly to how dietary lipids would have.
Among the dominant FAs, the proportions, i.e. relative amounts, of 14:0, 16:0, and thus that of SFAs, decreased most during the spawning run. Their proportions in salmon in the Baltic Proper were nearly as large as in salmon caught at the same location during their late autumn feeding migration [4]. During the spawning run, the proportions of 14:0, 16:0, and total SFAs were already significantly lower in salmon from the last sampling location in the sea in the Bothnian Bay than in the Baltic Proper. As all the sampled fish were females, SFAs were also used for the production of oocytes. According to Tocher [36], both SFAs and MUFAs, usually present in high percentages in storage triacylglycerols (TAGs), are heavily catabolised for energy, particularly in pre-spawning fish. In the present study, the decreases in the proportions of 18:1n-9 and total MUFAs were not prominent, probably because 18:1n-9 is an important constituent in both storage fat and structural phospholipids [36]. Thus, the utilisation of neutral lipids with a consequent relative increase in phospholipids did not greatly affect the 18:1n-9 proportion. All individual HUFAs were conserved more than other FAs, because they are characteristic of phospholipids. The consequent increase in the proportions of n-3 PUFAs and total PUFAs was greatly brought on by the quantitatively important DHA, which is a crucial component of the phospholipids of cellular membranes and is not easily used for energy production [36].
The increase in the proportion of DHA and n-3 HUFAs towards the spawning period is a general response among fish [36]. The proportion of DHA, contrary to its concentration, also increases during deprivation of food, as reported for sprat and herring [7]. In these species, the DHA proportions were higher in spring than in autumn and higher in the leaner specimens of the Gulf of Finland compared to the other Baltic Sea basins [7]. Moreover, HUFAs are the desaturation and elongation products of shorter PUFA precursors, although those enzyme activities are considered negligible in Atlantic salmon receiving HUFAs from their diet [36]. It is possible that the increase in the proportion of DHA partly results from some synthetic activity compensating for the cessation of dietary supply in pre-spawning fish [85], because DHA is still required for the synthesis of vitellogenin during the pre-spawning period [86]. However, considering the concentrations, i.e., the actual amounts, of FAs in muscle, they all decreased considerably by spawning time.
A major role of lipids in fish is to store energy as FAs and provide metabolic energy in the form of ATP through β-oxidation of FAs [36]. In feeding salmonine species, lipids largely accumulate in muscle tissue and in the body cavity as a visceral depot, the stores of which contain n-3 PUFAs in large percentages [6]. Although the proportion of PUFAs had increased by spawning, their actual amount had decreased by 46%, along with the use of lipids for metabolism and oocyte formation, during the spawning run and pre-spawning fasting. The hepatic concentration of MDA increased simultaneously, indicating large-scale peroxidation of PUFAs [63, 64]. Calculated from a linear model of the present study’s data, an increase of 1% in the muscular PUFA proportion of salmon was associated with an increase of 1.6 µmol g−1 in hepatic MDA concentration. In yolk-sac fry, the accumulation of MDA has been linked to M74 mortalities [87]. However, this has been linked to increased concentrations of DHA and n-3 PUFAs provided by females and an increased rate of lipid peroxidation [30, 56]. Since the MDA concentrations of the salmon at spawning were approximately double the concentrations detected in the salmon during the earlier phases of the spawning run, the oxidative stress caused by PUFA peroxidation was severe, although not fatal.
Manifestation of energy metabolism in total thiamine content of tissues
Although thiamine is water soluble, its halftime in non-breeding salmonines is fairly long. More than 10% of the original thiamine dose injected (i.p.) in juvenile steelhead trout [Oncorhynchus mykiss (Walbaum)] was detected in their muscle after six months [88]. In the present study’s salmon, from a year with an insignificant incidence of M74 [4], no clear trend in the concentration of the TotTHIA of muscle throughout the spawning migration was detected, although the concentrations at spawning were the smallest. Similarly, Karlsson et al. [89] found no decrease in the muscular thiamine concentration in the mid-1990s, when the incidence of M74 was high [4].
In hatchery salmon, the concentration of TotTHIA at spawning was, as in the present study, also largest in the liver; more than half this concentration was detected in the muscle, and one tenth in the blood [28]. In the present study, the decrease in the concentration of TotTHIA until spawning was clearer in the liver (> 60%) than in the other tissues studied, as in the study by Karlsson et al. [89], although their low sample number prevented statistical comparisons. In a year (1995) with high M74 mortality, Koski et al. [28] detected lower concentrations of total thiamine (3.3–6.6 nmol g−1) in the liver of spawning female salmon from the River Simojoki than we found for the salmon liver in the present study (6.3–10.3 nmol g−1). However, the values are probably not strictly comparable due to a different analysis method.
Although no significant decrease was detected in the concentration of TotTHIA in the muscle, contrary to the liver and ovaries, during the spawning run in the present study, the change in both the hepatic and muscular TotTHIA concentration was related to the decrease in the TotTHIA concentration of the eggs. In the single M74 female from the River Simojoki, whose offspring all died (while for all other females, offspring survival was > 94%), the TotTHIA concentration in both the eggs and muscle was the lowest measured in the present study. In a study by Amcoff et al. [14], the concentrations of thiamine in both ovaries and muscle were lower in those salmon whose offspring displayed M74 symptoms than in those females that produced normal yolk-sac fry.
Although no M74 mortalities were detected among offspring of salmon that had been feeding in the Baltic Proper, the concentration of TotTHIA in the ovaries of salmon decreased significantly during the sea phase of the spawning migration, and the ovulated eggs had only a quarter of the TotTHIA concentration of the ovaries from salmon sampled in the Baltic Proper. In the severe M74 years (1995–1997), caused by abundant feeding on young sprat in the Baltic Proper [4], the concentrations of thiamine in salmon liver and gonads tended to be lower in May than in April [89]. However, it is not very likely that the decrease in the concentrations of TotTHIA in the salmon liver and ovaries of the present study would have been clearer if the salmon from the Baltic Proper had been sampled earlier in the spring. In the salmon caught during the feeding migration in the same area in late autumn [Keinänen et al., unpubl.], the mean hepatic TotTHIA was only slightly larger than in the salmon from the present study.
Thiamine concentrations diminished as salmon decreased their food consumption and finally ceased feeding. Salmon had stopped feeding in the Bothnian Sea or at the latest when leaving it, approximately 500 km before entering the spawning river [18]. In addition, pre-spawning salmon fast until spawning in late October. The fasting period therefore lasts for about four months [18]. The more metabolic energy stored in lipids (as FAs need to be mobilised during the spawning run and pre-spawning fasting for the metabolic activities, such as acclimation to river water, the effort expended in swimming to the river’s spawning areas, and the ripening of oocytes), the more thiamine is depleted, because thiamine is required for energy metabolism [38]. However, high body lipid, and thus PUFA content of fish in general, has also been negatively correlated with thiamine concentrations during the feeding phase [7, 39].
Specifically, HUFAs, which are very susceptible to peroxidation [45, 46], cause an additional decrease in thiamine [51, 52]. Thiamine functions as a site-specific antioxidant in peroxidation reactions, in which it is converted to thiochrome and other forms, which can no longer act as the co-enzyme [47, 51, 52] and are excreted via the kidneys [38]. This process probably explains why the TotTHIA concentration decreased as a function of the increase in the concentration of DHA in sprat and herring and was lowest in the youngest specimens, in which the PUFA content was the highest [7]. As DHA, which among the FAs contains the highest number of double bonds, was the dominant PUFA during the spawning run, similarly to salmon during feeding migration [4], it was largely responsible for the depletion of thiamine in peroxidation reactions. According to a linear model, an increase of 1 mg g−1 in the concentration of DHA in the muscle resulted in a decrease of 2.4 nmol g−1 in the concentration of hepatic TotTHIA in the salmon sampled during the feeding migration in the autumn (Keinänen et al., unpubl.). Although oxidation of 18:1n-9 also increases oxidative stress, against which thiamine also acts as an antioxidant [47], the primary source of oxidative stress in fish tissues is peroxidation of HUFAs [45, 46]. As the content of n-3 HUFAs increases with the increase in the lipid content of fish, the tissues of the fattiest fish are most prone to lipid peroxidation [6, 43, 44] and thiamine deficiency.
Thiamine component composition in relation to lipid content
Although the TotTHIA concentration was highest in the liver, the thiamine of salmon is principally located in the muscle tissue, which is considerably more voluminous than the liver or ovaries. However, most (80–88%) of the TotTHIA in the muscle of salmon on their spawning run was detected as the co-enzyme form (TPP), similarly as has in general been detected for vertebrate soft tissues such as muscle, the brain, and the liver [38]. Muscular TPP probably does not easily supply additional thiamine for transporting to oocytes, because > 90% of the mitochondrial TPP in muscle is bound to enzymes [52]. The proportion of muscular TPP was higher in salmon after a decrease in feeding than in salmon caught at the first location of the spawning run in the Baltic Proper, and the TPP proportion increased as a function of the whole body lipid content of sprat and herring [7]. Apparently, in all these salmon, most of the thiamine was connected to metabolic processes as the TPP derivative. Muscular lipids were probably intensively turned over to metabolic energy and for the development of oocytes during pre-spawning fasting.
The median THIAM concentration in the muscle of salmon on the spawning run was very small, 0.070 nmol g−1, because most of the thiamine existed as TPP. The proportion of THIAM in muscle was 0.3–9.4%, which was lower than in most salmon caught during the feeding period in the autumn, except in the few fattiest ones among these salmon [Keinänen et al., unpubl.]. In the spawning salmon, the median concentration of THIAM in muscle was only 0.036 nmol g−1. The smallest concentration of THIAM in muscle and eggs was detected in the single M74 female from the River Simojoki. Being very small, the hepatic THIAM concentration changed very little during the spawning run, due to which it could not be used as an indication of thiamine status, contrary to the hepatic TotTHIA concentration.
M74 mortality and biochemical indices
Thiamine deficiency is manifested more easily in the yolk-sac fry than in their parents, because offspring, first as embryos and after hatching as yolk-sac fry, must rely on the thiamine reserves of yolk provided by the female parent for more than half a year during the rapid cell division and growth phase. In cases when adult salmon have a lower supply of thiamine than required for energy metabolism, taking into account thiamine depletion, adult salmon also suffer from thiamine deficiency. According to the Finnish M74 monitoring, all offspring of the females showing wriggling behaviour died due to thiamine deficiency [11].
All the offspring of one female in the present study succumbed to M74, whereas no M74 mortalities were recorded among the offspring of the five other females. The mean THIAM concentration (2.05 ± 0.22 nmol g−1) in the eggs from the present study’s River Simojoki salmon, excluding the single M74 female, was clearly larger than the limit value for any possible M74 occurrence. On the basis of long-term Finnish M74 monitoring [4, 11], at an egg THIAM concentration of ≤ 0.2 nmol g−1, yolk-sac fry mortality of 100% is expected, and at a concentration of ≤ 0.5 nmol g−1, the female is very probably a M74 female, whereas at the THIAM concentration of eggs ≥ 1.0 nmol g−1, possible mortalities are not caused by thiamine deficiency. The mean THIAM concentration in the eggs of the non-M74 females in the River Simojoki from the present study was also considerably larger than the median concentration of 0.35 nmol g−1 in the eggs of the salmon included in the Finnish M74 monitoring in 1994–1995, when the incidence of M74 was very high [4]. However, due to the variation in the feeding areas of salmon, and thus in their dietary composition and abundance [1, 3, 33], the egg THIAM concentrations of individual salmon included in the Finnish M74 monitoring have varied greatly in some years, even more than a 100-fold in a year [4]. In four salmonine species from Lake Ontario, the LC50 values of free thiamine in their eggs varied from 0.26 to 1.32 nmol g−1 [2]. Although the mean THIAM concentrations of eggs in this study’s non-M74 females had not decreased alarmingly when metabolising lipids and peroxidation-susceptible PUFAs, the salmon may have been physiologically stressed, as has been reported for humans and other vertebrates with a reduced thiamine status [52].
In addition to the decreased concentrations of TotTHIA and its components in the liver and ovaries by spawning, the risk of developing oxidative stress also became apparent in these spawning salmon in their reduced muscular carotenoid concentrations. Carotenoids have also been transported from the muscle into developing oocytes during exogenous vitellogenesis. Small carotenoid concentrations in the eggs of M74 female and a link between M74 and oxidative stress have been found in earlier studies [29, 30, 56] and in the Finnish M74 monitoring [11]. The smallest carotenoid concentrations in the muscle were detected in the single M74 female and in one non-M74 female. Thiamine deficiency in itself is known to increase oxidative stress [51, 90], and carotenoids as antioxidants are therefore consumed. Contrary to the thiamine concentration, no decrease in the carotenoid concentration was found during the marine phase of the spawning run. In addition, the carotenoid concentration had no significant relationships with the concentrations of TotTHIA in any tissue studied during the spawning run. As carotenoids are powerful general antioxidants [59, 91], they are depleted due to oxidative stress caused by various factors (e.g. environmental toxicants [92]), whereas thiamine functions as an antioxidant against lipid peroxidation products [47, 51, 52]. The muscular carotenoid concentrations appeared to vary greatly among the different sampling locations during the spawning run. As salmon obtain astaxanthin from the crustaceans in the alimentary tract of sprat and herring [93], the dietary condition of prey fish directly affects salmon astaxanthin concentration. In their turn, the astaxanthin production by Baltic Sea mesozooplankton is affected by changes in environmental temperature and salinity, for example [94].
During the spawning run, there was a large variation in hepatic EROD activity, but in spawning salmon, the EROD activity was significantly lower than in salmon caught in the sea. This was unexpected, because organohalogens are known to induce EROD activity [71], and the concentrations of organohalogens in the muscle of the present study’s salmon had increased by spawning [18]. The salmon’s organohalogen burden had barely changed, but as a consequence of hydrolysing body lipids for metabolism and transferring them to the developing oocytes, the xenobiotic concentration per body mass increased [18]. However, it was reported that the concentrations of PCBs, DDTs, and polybrominated diphenyl ethers did not differ between the M74 females and non-M74 females [95]. Likewise, the thiamine deficiency of lake trout [Salvelinus namaycush (Walbaum)] in the Great Lakes had no relationship with the concentrations of organochlorines in their eggs [96]. However, oestrogens, whose concentrations maximise before spawning, are known to suppress EROD activity [97, 98], and some PUFAs (e.g. DHA) have also been demonstrated to suppress it [99]. Vitellogenin, which is synthesised in the liver, consists of approximately 20% of DHA [86]. Thus, high PUFA contents may have for their part impaired hepatic EROD functioning in metabolising oestrogens and organohalogens, the latter of which were translocated in the turnover of tissue energy stores during pre-spawning fasting.
Thiamine status of salmon in relation to their feeding conditions
It is possible that the salmon caught in the different sampling locations included individuals from rivers that do not run into the Bothnian Bay. However, all the salmon sampled at spawning had apparently returned to their home river, because they were caught at the mouth of the River Simojoki. In any case, most of the sampled salmon had been feeding in the Baltic Proper. The only exceptions were one salmon from the Bothnian Bay and the single M74 female which ascended the River Simojoki, both of which had probably been feeding in the Bothnian Sea. In all the other salmon, the ratio of the total concentration of PCDD/F to the concentration of CoPCB and the muscle FA signatures were characteristic of those salmon feeding in the Baltic Proper [4, 18, 66].
In the Baltic Proper, salmon mainly prey on sprat [3, 17], which was also found by analysing the stomach content of the present study’s salmon [18]. Those salmon caught along the route of the spawning run may have been feeding in various sub-areas of the Baltic Proper, which differ in relation to the proportions of sprat and herring in a salmon prey biomass [3, 33]. This caused the variation seen in the THIAM concentrations in the eggs of non-M74 females, for example. According to Jacobson et al. [33], the dietary proportions and size of sprat and herring also appeared to depend on the size of salmon, but in the present study, all the salmon had spent two years in the sea and were of the same size.
Salmon prefer small prey [17, 18]. As herring grow fastest in the southernmost parts of the Baltic Sea, only the youngest age groups of herring in the area are of the right size to be preyed on by salmon [3]. In contrast, all sprat are of a suitable size for salmon [3]. In those years with a high or moderate incidence of M74, mortalities were regarded as a consequence of feeding abundantly on young sprat in the Baltic Proper [1, 3]. Feeding principally on sprat in the southern Baltic in 1998, when there was a moderate incidence of M74, was also verified by FA signature analysis [4].
Between 2002 and 2004, when the salmon from the present study were feeding in the Baltic Proper, the sprat stock had declined to its smallest size after its explosive increase at the turn of the 1990s [11], and the year classes for sprat in these years were especially small [100]. All the River Simojoki salmon at spawning addressed in this study (excluding the single M74 female) had been feeding in the Baltic Proper and mainly on sprat, indicated by the high 18:1n-9/ARA and 14:0/ARA ratios [4]. Apparently, these salmon had largely fed on older sprat specimens whose lipid content was lower, and whose thiamine concentration was higher than in young sprat [1, 7], because the THIAM concentrations in their eggs were higher than the estimated limit value for M74 symptoms. However, the THIAM concentration in eggs from the River Simojoki salmon had decreased to half the THIAM concentration in the ovaries of salmon from the Baltic Proper. Consequently, their thiamine resources had depleted during the spawning run and pre-spawning fasting, but to a lesser extent than in those cases that had resulted in M74.
Because thiamine concentrations decreased after the salmon diminished and ceased feeding, the thiaminase of prey fish cannot be the cause of thiamine deficiency in salmon eggs and tissues, contrary to an old and long-lived hypothesis, as has already been demonstrated in detail by Mikkonen et al. [3] and Keinänen et al. [1]. As discussed by Keinänen et al. [4], the large size and high growth rate of salmonines whose offspring suffer from thiamine deficiency also verifies that the thiamine supply from prey fish has been high enough to enable proper growth. The growth rate of salmon has been proper, ranging from 20 to 32 cm per year from the first to second growth seasons during the period 1971–2006 [1, 3]. Growth was faster during the moderate or high incidence of M74 in the 1990s and in the 2000s than that from the mid-1970s to the early 1980s, when M74 mortalities were insignificant [3, 26]. As retarded growth is a symptom of dietary thiamine deficiency [38], the thiamine reserves in salmon at the start of the spawning run in the Baltic Proper, even during the years of high M74 incidence, have apparently been adequate considering the requirement for their proper growth [40]. Indeed, there are no publications that could have proven the linkage of thiaminase in prey fish to thiamine deficiency in salmonines, despite the fact that the topic has been greatly studied, with dozens of publications concerning it. By contrast, an increased requirement of thiamine with an increase in the dietary energy content is well known for humans, as well as for fish [40, 41]. Likewise, the depletion of thiamine in energy metabolism and peroxidation reactions in the tissues of vertebrates is well documented [41, 51, 52]. In all our studies concerning M74 and in our reviews of publications concerning other thiamine deficiency syndromes of salmonines (e.g. TDC), thiamine deficiency is consistent with such a cause [1, 3, 4, 7]. As the accumulation of lipids and n-3 PUFAs in fatty fish species increases with an increase in the lipid and n-3 PUFA content of a fish-based diet [6, 44], extreme circumstances can result in serious thiamine deficiency in salmonines, as was found in the years with the highest incidence of M74 during the 1990s [4, 11].
The proportion of sprat in general decreases from the southern to the northern Baltic, meaning that in the Bothnian Sea, herring is the dominant species [17, 26]. Sprat shoals, however, disperse from their spawning areas of the Baltic Proper, particularly when the sprat stock has increased following the years of their very successful reproduction [32]. Practically no sprat have been caught from the most northern part of the Gulf of Bothnia, the Bothnian Bay [100]. Between 2002 and 2003, age 0–1 herring were abundant as prey for salmon in the Bothnian Sea, as these year classes were very strong [100]. Age 0 herring during the late summer already attain lengths that are appropriate for them to be prey for salmon [17, 101]. In the Bothnian Sea, only approximately 14% of the salmon from the River Simojoki stock halt to feed [34], based on a long-term average. The concentration of DHA and total PUFAs in the youngest specimens of herring, similarly to sprat, are the highest [7]. Moreover, the proportion of PUFAs was or tended to be on average higher in herring than in sprat [7, 80]. In salmon feeding between 2002 and 2003 in the Bothnian Sea, the proportion of DHA, and consequently that of n-3 PUFAs and total PUFAs, was higher than in salmon caught from the Baltic Proper [4]. Thus, it is possible that PUFAs obtained by feeding on young fatty herring in abundance enabled the sporadic development of M74 in a year when feeding in the Baltic Proper did not result in thiamine deficiency [4].