Female and male EB cod were pooled (A-C). The colors correspond to the age of the specimen, determined by annuli counting of the otoliths. (A) The otolith weight increased with increasing age determined by annuli counting of the otoliths. (B) The brain weight increased with increasing otolith weight. (C) The somatic weight increased with increasing otolith weight. (D) The female gonad weight increased with increasing otolith weight.

The age by annuli counting of the EB cod is difficult to determine [ 76 ]. Different variables were plotted against each other to find the best variable corresponding to the age by annuli counting. The otolith weight correlated strongly with the age by annuli counting (P<0.0001, n = 50, Fig 4A ), a phenomenon observed among many teleost fish species [ 77 – 82 ]. The growth of the brain is one of the most stable variables for an animal suffering from thiamine deficiency [ 83 ]. The growth of the brain can be considered relatively constant as an allometric standard representative for the age of the cod ( Fig 4B ). The brain weight increased with increasing age (P<0.0001, n = 50, represented by colors in Fig 4B ) and with increasing otolith weight (P<0.0001, n = 50, Fig 4B ). The somatic weight increased with increasing age (P<0.0001, n = 50, represented by colors in Fig 4C ) and with the otolith weight (P<0.0001, n = 50, Fig 4C ). It can be assumed that the best way to estimate the age of the cod in this study was through the brain weight or the otolith weight. According to previous studies regarding otolith weight from cod, the cod in this study were assumed to range in age from 1–6 years old [ 82 ]. The age by annuli counting of the cod corresponded to exactly the same range, i.e. 1–6 years old ( Fig 4A ). The female gonad weight increased with increasing age (P<0.0001, n = 29, represented by colors in Fig 4D ) and with the otolith weight (P<0.0001, n = 29, Fig 4D ). There were a few female specimens that did not follow the trend of increasing the gonad weight as the otolith weight increases. It could be that these specimens are not able to mature properly, however further investigations more closely to the spawning time period should be made before conclusions can be drawn. There was no correlation between the LSI and gonad somatic index in females (P = 0.88, n = 29, not shown), indicating that the gonad has not started to mature.

Age was determined with annuli counting of the otolith. The total length of the specimen is presented in the brackets (A) A cod with CF = 0.95, age 6 years (B) A cod with CF = 0.84, age 2 years (C) The cod with the lowest CF = 0.61, age 3 years.

The studied EB cod group, consisted of 51 specimens caught at different sites in Hanöbukten Bay during late October and early November 2017. In the studied EB cod group, 57% were female, 35% were male and the sex of 8% of the specimens were visually unidentifiable. The mean length of the specimens was 39.3 cm (range 23.1–56.5 cm). The mean total weight was 529 g (range 110–1530 g). The mean somatic weight was 522 g (range 110–1510 g). The mean Fulton’s condition factor was 0.803 (range 0.605–1.05). The mean somatic condition factor was 0.793 (range 0.589–1.04). The mean liver somatic index (LSI) was 3.71% (range 1.15–6.89%). The mean liver weight was 22.1 g (range 1.80–104 g). The mean brain weight was 0.720 g (range 0.310–1.20 g). The average weight of the two otoliths was 232 mg (range 65.0–399 mg). The average otholith weight was used for the data analysis. The mean age by annuli counting was 3.8 years (range 1–6 years). The mean quality of the annuli counting was 1.9 (range 1–3). Condition indices were consistently low in this study and were comparable to previously observed values in the Baltic Sea. Between 1987 and 1996, the EB cod CF ranged from 1.1–1.2 in the same area [ 75 ], values that may be considered as normal healthy control values. In our study, only 2% of the EB cod sampled had a CF above 1 ( Fig 2 ). A previous study has shown that CF values below 0.9 from EB cod from the Bornholm Basin were outside the standard deviation in 2000 [ 46 ]. The current study found that 76.5% of the cod had a CF value below 0.9. Furthermore, 49% of the studied group had a CF value below 0.8. This can be compared to less than 30% of cod with a CF value below 0.8 in 2014 [ 46 ]. The specimens in this study are small and ill-proportioned ( Fig 3 ), in which specimens with different CF values are shown. Most of the biological data were acquired for all specimens (51), except for otoliths where one was missing (50), and thiamine data, where biochemical measurements were done on 30 liver samples and 22 brain samples, and chemical measurements were done on 38 liver samples.

Evidence of severe thiamine deficiency in liver and brain

The concentration of liver SumT decreased as liver size increased (P<0.0001, n = 38, Fig 5A). The SumT decreased with increasing age (P = 0.017, n = 38, as shown in Fig 5A). The liver weight increased with increasing age (P<0.0001, n = 50, represented by colors in Fig 5A). The concentration of liver SumT decreased with increasing relative size of the liver, LSI (P<0.0001, n = 38, Fig 5B). The LSI increased with increasing age (P = 0.0041, n = 50, represented by colors in Fig 5B).

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larger image TIFF original image Download: Fig 5. Relationship between liver SumT concentration and liver weight. The colors correspond to the age of each specimen, determined by annuli counting of the otoliths. (A) The concentration of T, TMP and TDP (combined SumT) decreased with increasing liver weight (Spearman correlation). (B) The SumT concentration decreased with increasing relative liver somatic weight (LSI). https://doi.org/10.1371/journal.pone.0227201.g005

The low concentration of TDP in the liver in many specimens is reflected in the strong correlation between a decrease of TK activity and increased proportion of TK apoenzymes in the liver, i.e. high latency (P = 0.0028, n = 30, Fig 6A). This is a known correlation observed both among laboratory experimental animals and among wild populations with thiamine deficiency [16, 32, 84]. When the concentration of SumT is low, the proportion of apoenzymes is high. However, the apoenzymes will degrade due to instability [85]. The activity might not be as high as it previously was in a specimen that has suffered from thiamine deficiency for a long period of time [85]. There was a tendency of a correlation between decreased liver TK activity and increasing age (P = 0.074, n = 30, represented by colors in Fig 6A) and no correlation between liver TK latency and age (P = 0.23, n = 30, represented by colors in Fig 6A). In this study, the correlation between the TK latency and activity is stronger in the brain (P<0.0001, n = 22, Fig 6B) compared to the liver (Fig 6A). This is most likely due to better protection against degradation of the apoenzymes in the brain than in the liver, which has been observed previously [86]. Important to note, however, this does not mean that the brain is more thiamine deficient. Due to the fact that the brain is better protected, and thus affected later in the deficiency, no correlation could be seen between increasing age and decreasing activity (P = 0.4068, n = 22, represented by colors in Fig 6B) or increasing latency (P = 0.3148, n = 22, represented by colors in Fig 6B).

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larger image TIFF original image Download: Fig 6. Relationship between TK specific activity and latency, in liver and brain tissue in EB cod. The colors correspond to the age of each specimen, determined by annuli counting of the otoliths. (A) The liver TK activity decreased with increasing liver TK latency. (B) The brain TK activity decreased with increasing brain TK latency. https://doi.org/10.1371/journal.pone.0227201.g006

The proportion of the different forms of thiamine can reflect the thiamine status in a tissue. The proportion of T, TMP and TDP were compared between the 15 specimens with the lowest Sum T concentrations (group A) and the 15 specimens with the highest SumT concentrations (group B) (Fig 7). The distribution of the thiamine forms in the lower concentration of SumT, group A (<8 nmol SumT/g liver), was 2.9% T, 81% TDP and 16% TMP while group B (>11 nmol SumT/g liver) had 3.5% T, 77% TDP and 20% TMP. There was no difference between the proportion of T between the groups (P = 0.16). However, the proportion of TDP was higher in group A than in group B (P = 0.0019). The proportion of TMP was lower in group A than in group B (P = 0.0035). It is expected that at low SumT concentrations, the proportion of TDP is kept high to maintain thiamine dependent metabolism, and consequently the proportion of T and TMP are lower than when SumT concentrations are normal [16, 73]. The low concentration of T suggests a normal function of the thiamine pyrophosphokinase, since a thiamine deficient specimen can keep a high proportion of TDP in the cells, a phenomenon that has been previously observed among several wild animals with thiamine deficiency [32]. This indicates that the explanation for thiamine deficiency is not likely due to a malfunction of the enzyme thiamine pyrophosphokinase [32].

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larger image TIFF original image Download: Fig 7. Proportion of T, TDP and TMP in the 15 specimens with the lowest and highest SumT concentrations. The distribution of T, TMP and TDP among the 15 specimens with the lowest SumT concentrations (group A), to the left and the 15 specimens with the highest SumT concentrations (group B), to the right. The proportion of T did not differ between the groups. The proportion of TDP was higher in group A than in group B (P = 0.0019). The proportion of TMP was lower in group A than in group B (P = 0.0035). Error bars correspond to a 95% confidence interval. https://doi.org/10.1371/journal.pone.0227201.g007

When the somatic weight increased, the liver TK latency increased (P = 0.017, n = 30, not shown). When the length of the EB cod increased, the liver TK latency increased (P = 0.025, n = 30, not shown). As the liver size increased, the liver TK latency increased as well (P = 0.049, n = 30, not shown). When the relative weight of the brain increased, as typically seen in younger fish, the liver TK latency decreased (P = 0.021, n = 30, not shown). As the otolith weight increased, the endogeneous TK activity in the brain decreased (P = 0.0062, n = 10, blue line Fig 8A), while the maximum TK activity seems to be relatively constant (P = 0.64, n = 10, red line Fig 8A). As the otolith weight increased, the brain TK latency in males increased (P = 0.023, n = 10, Fig 8B). There was no correlation between the liver TK latency and the otolith weight, a phenomenon that may be based on higher degradation of the apoenzymes in the liver tissue.

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larger image TIFF original image Download: Fig 8. Relationship between otolith weight and TK variables in the brain. (A) The TK endogeneous activity (blue) in the brain decreased with increasing otolith weight while the TK maximum activity (red) was relatively constant with increasing otolith weight. (B) The colors correspond to the age by annuli counting of the otoliths. The brain TK latency increased with increasing otolith weight. Two specimens had latency values below zero (otolith weights 138 mg and 213 mg) and were therefore excluded from the graph, but included in the statistical calculations. https://doi.org/10.1371/journal.pone.0227201.g008

There was no correlation between the weight of the brain and the liver TK latency. However, by excluding brains smaller than 0.60 g, mainly corresponding to EB cod younger than 3 years, the liver TK latency increased with increasing brain weight (P = 0.0062, n = 22, Fig 9A). The liver SumT concentration decreased with increasing otolith weight (P = 0.0031, n = 37, Fig 9B), as well as with the age by annuli counting of the otoliths (P = 0.017, n = 37, represented by colors in Fig 9B). This indicates that the thiamine deficiency develops as EB cod get older. Higher demand of thiamine for reproduction and/or lower uptake of thiamine from the food chain cannot be excluded as explanations, given our present state of knowledge.

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larger image TIFF original image Download: Fig 9. Relationship between thiamine biomarkers and biological parameters. The colors correspond to the age (annuli counting) of each specimen. (A) The liver TK latency increased with increasing weight of the brain when excluding brains smaller than 0.6 g. (B) The liver SumT concentration decreased with increasing otolith weight and age by annuli counting of the otoliths. https://doi.org/10.1371/journal.pone.0227201.g009

Thiamine and its derivatives decreased significantly with increasing otolith weight; T (P = 0.025, n = 37), TMP (P = 0.0018, n = 37) and TDP (P = 0.0041, n = 37) (Fig 10A–10C). The concentration of thiamine and its derivatives decreased with decreasing SumT, T (P<0.0001, n = 37), TMP (P<0.0001, n = 37) and TDP (P<0.0001, n = 37) (represented by colors in Fig 10A–10C). Previous studies have shown that the proportion of TDP increased with severity of thiamine deficiency in specimens with thiamine deficiency [32]. The proportion of liver TDP increased with increasing otolith weight (P = 0.029, n = 37, Fig 10D) and increasing total weight (P = 0.0095, n = 38, not shown). The proportion of TDP increased with a tendency of significance with the age (P = 0.056, n = 37, represented by colors in Fig 10D). This correlation indicates that, as a result of a lower thiamine status, larger and older specimens have the highest proportion of TDP.

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larger image TIFF original image Download: Fig 10. Thiamine concentrations and proportions in the studied EB cod group. Thiamine (T), thiamine monophosphate (TMP) and thiamine diphosphate (TDP) are combind to SumT. (A) The liver T concentration decreased with increasing otolith weight. The colors correspond to the concentration of SumT. (B) The liver TMP concentration decreased with increasing otolith weight. The colors correspond to the the concentration of SumT. (C) The liver TDP concentration decreased with increasing otolith weight. The colors correspond to the concentration of SumT. (D) The proportion TDP (percentage of liver SumT) increased with increasing otolith weight. The colors correspond to the age of each specimen, determined by annuli counting. (E) The proportion TMP (percentage of liver SumT) decreased with increasing otolith weight. The colors correspond to the age of each specimen, determined by annuli counting. (F) The proportion TDP (percentage of liver SumT) decreased with increasing liver TMP concentration. The colors correspond to the concentration of SumT. https://doi.org/10.1371/journal.pone.0227201.g010

The proportion of liver TMP decreased with increasing otolith weight (P = 0.013, n = 37, Fig 10E) and increasing total weight (P = 0.0033, n = 38, not shown). The proportion of TMP decreased with increasing age (P = 0.024, n = 37, represented by colors in Fig 10E), indicating that older specimens might be more thiamine deficient. In a thiamine deficient specimen, there is usually an increase in the proportion of TDP as the TMP concentration decreases [32]. One explanation for this is that the intracellular utilization of TMP increases in specimens with severe thiamine deficiency [32, 87, 88]. This correlation is observed within this EB cod group, the proportion of liver TDP increased with decreasing liver TMP (P = 0.00016, n = 38, Fig 10F). The proportion of liver TDP decreased with increasing SumT (P = 0.028, n = 38, represented by colors in Fig 10F).

The liver TK latency increased as the liver weight increased (P = 0.049, n = 30, not shown), correlating with the chemical analysis (Fig 5A). The correlation could depend on the total weight and/or the age of the EB cod, where older EB cod are more thiamine deficient than younger EB cod. The relative size of the liver does not affect the liver TK latency (P = 0.5029, n = 30, not shown). The brain TK latency increased as the SCI in females increased (P = 0.00084, n = 12, not shown). In previous studies, the latency decreases as the SCI increases [32]. However, among these EB cod specimens in this correlation between SCI in females and brain TK latency, 83% had a SCI below 0.8, indicating that the thiamine dependent apoenzymes are unstable when the deficiency has prolonged to cause drastic changes to the body condition [85].

The mean liver TK latency was 15 ± 4.5% (range 0–49%, n = 30). The mean brain TK latency was 27 ± 8.3% (range 0–66%, n = 22). The mean liver SumT concentration was 10 ± 1.9 nmol/g (range 2.4–24 nmol/g, n = 38). Due to the instability of the TK apoenzymes in the liver, it is difficult to determine a specific SumT concentration where the EB cod is above the threshold for thiamine deficiency. By combining the chemical and the biochemical analysis, it can be assumed that in a healthy EB cod specimen the SumT concentrations in the liver should at least be in the region of 20 nmol/g or higher. There are many specimens with a concentration half of that, suggesting severe thiamine deficiency in this group. Furthermore, previous analyses of EB cod collected in 1996 showed that female EB cod had liver SumT concentrations below 2 nmol/g and male EB cod had liver SumT concentrations below 4 nmol/g [33], indicating thiamine deficiency in this population more than 20 years ago. These results, combined with the long-term declines seen in EB cod over the past 30 years, raises the question whether the collapse of the EB cod population in the Baltic sea is related to thiamine deficiency. The EB cod seem to be in an even worse state than the Atlantic salmon (S. salar) and European eel (A. anguilla), with individual specimens with higher latency in both liver and brain, see Table 1, in addition to the very low levels of SumT in older specimens (Fig 9B).

The analyzed liver tissue showed that 76% of the EB cod had thiamine deficiency in the liver (Fig 11A) with an average of 19% liver TK latency (n = 23, not shown) among the thiamine deficient specimens. The analyzed brain tissue showed that 78% of the EB cod had thiamine deficiency in the brain (Fig 11B) with an average of 34% brain TK latency (n = 17, not shown) among the thiamine deficient specimens. These measurements showed that 13.3% of the livers and 63.6% of the brains where severely thiamine deficient (latency >25%) (Fig 11A and 11B). At first sight, this could give the impression that the brain is more affected than the liver of the ongoing thiamine deficiency. However, our interpretation is that the TK apoenzyme might be more stable in the brain tissue than in the liver tissue, resulting in higher latency values in the brain (Fig 6A and 6B). Even though this study only measured the SumT concentration in the liver, the fact that a decline of SumT is often more pronounced in the liver than in the brain, is an established phenomenon among different species where thiamine deficiency has developed over time [17, 16, 32, 89, 90].

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larger image TIFF original image Download: Fig 11. Proportion of the EB cod with no thiamine deficiency, thiamine deficiency and severe thiamine deficiency in liver and brain tissue. Based on an arbitrary definition, non-thiamine deficient tissues were defined as tissues with TK latency <6%, thiamine deficient tissues were defined as tissues with 6–25% latency, and severely thiamine deficient tissues were defined as tissues with more than 25% latency. (A) The liver showed obvious thiamine deficiency in 63% and severe thiamine deficiency in 13% of the EB cod. (B) The brain showed obvious thiamine deficiency in 14% and severe thiamine deficiency in 64% of the EB cod. https://doi.org/10.1371/journal.pone.0227201.g011

The summarized results from this study are compared with European eel, Atlantic salmon [32] and results from the previous study in 1996 [33] regarding EB cod in Table 1. In fact, among the EB cod in this study, there were a few specimens that had up to ten times lower SumT concentrations compared to others, also suggesting that the analyzed EB cod group contains specimens that have severe thiamine deficiency.

A declining cod population was observed in Newfoundland in the 1980s, and in 1992 all commercial fishing of Atlantic cod in the area was banned [91]. The explanations for the disappearance of the Atlantic cod in that area was over-fishing, the same as one of the major hypotheses for the decline in the Baltic Sea today. However, the Newfoundland Atlantic cod population has not recovered since then, despite large reductions of fishing pressure [92]. Other observations included smaller fish [93], early maturation [93, 94], lower body condition [95, 96], decline of energy reserves [96] and skipped spawning [97]. The cod population in Newfoundland had a decreasing CF just like the EB cod population today [98]. The fact that the population has not recovered has led to a change in speculation for the decline, to "elevated natural mortality" [94]. However, these symptoms are not inconsistent with thiamine deficiency, and appear similar to what we see currently happening to the EB cod. To our knowledge, the concentration of thiamine and the TDP-dependent enzymes has not been investigated in the Atlantic cod population in the waters outside Newfoundland, and it cannot be ruled out that the population might suffer from thiamine deficiency in this region. In 1958, the average CF in the Newfoundland cod population was varying around a mean of 1, and decreased to around 0.85 in 1993 [95]. It seems that the cod in Newfoundland die around a CF value of 0.4 [95], and this could explain the lack of specimens below 0.6 in our study. Toxic compounds such as glyoxals and lactic acid may reach lethal values in these species [18, 19]. The liver SumT concentration in the EB cod population could be assumed to be lower in adult tissues in connection with gonad development and egg maturation. Because this study sampled EB cod about 6 months prior to maturation of the eggs, lower levels of thiamine in the adult tissues could be expected closer to the spawning period. In fact, this difference in sampling period compared to the previous study in 1999 may at least partly explain their even lower SumT levels in the liver [33]. While compiling results from across years suggests that the wild EB cod population is thiamine deficient during the entire year, greater temporal resolution is required to determine whether this might be the case.

Results in 1994 indicated that the reproductive success of EB cod was impaired, and that there were increases in mortality and disorders among the offspring correlated to the female, similar to the effects seen in the offspring of thiamine deficient Baltic salmon [15]. A previous study in 1999 argued that the reproductive failure and population decline of the EB cod was not due to M74 [99], which is an old, partly misleading term for thiamine deficiency in salmon offspring [32]. However, the conclusion was drawn based on the comparison of the concentration of thiamine in eggs in Atlantic salmon, incorrectly assumed healthy, compared to the EB cod gonad concentrations [33]. Today we know that these SumT concentrations were too low to produce healthy offspring [32]. In fact, the authors from the work in 1999, who performed the chemical analytical work, concluded that they were not able to determine the thiamine status in EB cod at the time of publication [33]. Fish are affected by thiamine deficiency during embryonic and larval development, because the thiamine deficient adult female is not able to transport the necessary amount of thiamine to the maturating eggs [100]. Larvae with a low thiamine level can therefore be assumed to die in the early life stages. The adult EB cod are affected by thiamine deficiency, and might die directly, as a result of glyoxals, lactic acid, phytanic acid and/or neurological disturbances. However, death probably more commonly occurs as a consequence of secondary disorders of the deficiency, such as orientation problems, weakened senses and/or immunosuppression leading to infections of bacteria, virus, fungi and/or parasites [32]. Previous studies have shown that certain animals become anorectic and emaciated during thiamine deficiency [16]. Furthermore, it has also been shown that starvation does not lead to an increase in latency, simply due to the fact that when a specimen is starving, it does not eat, and does not need to metabolize any food, and therefore the specimen needs less thiamine [16, 84]. Thus, the fact that the CFs of the EB cod are low, cannot explain the thiamine deficiency, although, the thiamine deficiency could explain the low CFs.

The increase of the infestations and prevalence of nematodes in the EB cod could be due to a thiamine deficiency leading to immunosuppression. For example, rats with thiamine deficiency are more susceptible to different parasitic infections, including nematode infections [101]. Furthermore, the thiamine deficient European silver eels and American eels, are infected by the nematode Anguillicola crassus, and the prevalence has increased throughout the assumed time period of thiamine deficiency and during the species decline in the last decades [32].

As a final remark, the low growth, low body condition, high mortality, altered metabolism, survival of offspring, emaciation and parasite infections of the EB cod, stated in the recently published ICES report [43], are all common and expected symptoms of the thiamine deficiency that is present among the EB cod.