DOI: 10.5061/dryad.cjsxksnjc
title: Thiamine allocation and deficiency status throughout the life cycle of cod

description: Several wild bird and fish species across the Northern Hemisphere have
 been shown to episodically be thiamine (vitamin B1) deficient, leading to
 mass-mortality events in offspring. To understand the mechanisms
 underlying thiamine deficiency, we need a better understanding of the
 dynamics and somatic allocation of the vitamin. Here, we focus on an
 ecologically and economically important species, i.e., Atlantic cod (Gadus
 morhua), which has been suggested to be sensitive to thiamine deficiency.
 We sampled cod of varying sizes and maturity stages in a system where
 thiamine deficiency regularly occurs (i.e. Baltic Sea) and compare these
 with cod from the North Atlantic, where deficiency has not been recorded.
 Results show that thiamine concentrations were tissue-specific.
 Concentrations in muscle and liver declined during growth and maturation,
 whereas concentrations in gonads increased. Of the total thiamine in a
 female’s body, approximately 70% was allocated to the gonads at the onset
 of reproduction, suggesting that micronutrients constitute a major
 investment when spawning. Transketolase activity suggest that livers were
 saturated with thiamine and there was no evidence of ongoing thiamine
 deficiency. We show that life cycle and tissue-specific dynamics in
 thiamine concentrations should be considered when assessing the thiamine
 status of a species.
description: Study system & sampling Cod specimens
 were sampled in the North Sea, Baltic Proper, and Åland Sea (Fig. 1A). In
 the North Sea, thiamine deficiency has never been observed, and while cod
 stocks are declining, cod still show good condition and grow to large
 sizes (Fig. 1B,C). This is also true for cod in the Åland Sea, whereas
 most other stocks in the Baltic Sea, including the Baltic Proper, have
 shown rapid declines, low condition factors, and size truncation. Hence,
 we used the North Sea and Åland Sea stocks as reference points to compare
 them to the Baltic Proper stock, which is suffering from the
 aforementioned symptoms and a suggested thiamine deficiency. Through
 coalescing the data on thiamine from all three stocks, we seek to better
 understand the extent of the potential thiamine deficiency as well as the
 general thiamine dynamics throughout cod development and
 reproduction. All sampled cod individuals were caught
 as part of environmental monitoring programs conducted by the Swedish
 University of Agricultural Sciences, Department of Aquatic Science (SLU
 Aqua). A total of 193 specimens (<em>n</em> = 193) were
 sampled. In the North Sea, 82 individuals were collected through bottom
 trawling between January and February 2022. Similarly, in the Baltic
 Proper, 91 individuals were caught between February and March 2022.
 Detailed information about these expeditions can be found in the cruise
 reports (Bland and Börjesson 2022, Lövgren and Casini 2022). In the Åland
 Sea, 20 cod were obtained through cooperation with a local fisherman using
 stationary deep-water nets that were placed overnight during June 2022
 (Heimbrand et al. 2023). For each cod, the weight,
 length, and sex (if possible) were recorded. The maturity level was
 estimated categorically following standardized protocols (Tomkiewicz et
 al. 2002). For simplicity, we combined categorical maturity stages into
 three major maturational stages: Preparation (1,2), maturation (3,4), and
 spawning (5-7). Liver, gonad, and gastrointestinal tract were separated
 and weighed to the nearest gram. Where possible, approximately 20 g of
 dorsal muscle, liver, and gonad tissue were vacuum-packed and stored at
 -80°C for thiamine and transketolase analysis (see below). In cases where
 the liver or gonad weighed less than 5 g, it was weighed again on land to
 an accuracy of 0.001 g. Not all individuals caught during the
 environmental monitoring programs were sampled for this study. We selected
 individuals from as many size classes as possible to ensure a
 representative sample of the stocks’ demography. To control for putative
 variation within stocks and/or environments, we tried to sample specimens
 from as many sites as possible. Individuals smaller than 14 cm were
 excluded from the study as their intestinal mass was insufficient for
 thiamine analysis. Thiamine analysis
 Thiamine was analyzed in muscle, liver, and female gonad tissues
 according to Brown et al. (1998) with minor modifications. For a detailed
 description of the process see Todisco et al. (2024). In short, tissues
 were homogenized and boiled in diluted trichloroacetic acid. The
 supernatant was washed using a mixture of ethyl acetate and hexane before
 adding K<sub>3</sub>Fe(CN)<sub>6</sub> as a dye.
 Samples were analysed in a Hitachi Chromaster HPLC system by measuring
 fluorescence. Three vitamers of thiamine were quantified: free thiamine
 (TF), thiamine monophosphate (TMP), and thiamine diphosphate (TDP).
 Concentrations (unit: nmol/g) were normalized for wet and dry weight (see
 below) and combined to total thiamine (Ttot).
 Transketolase We selected 48 female cod
 specimens which covered the total size range and liver thiamine
 concentrations to measure their transketolase activity and latency. To
 compare the different stocks, we selected 18, 17, and 13 specimens from
 the North Sea, Baltic Proper, and Åland Sea, respectively. The activity
 and latency of transketolase was measured in liver tissues using the BCA
 Protein Assay Kit (ab102536, Abcam; prev. K813-2500/5000, BioVision) and
 Transketolase Activity Assay Kit (ab273310, Abcam; prev. K2004-100,
 BioVision). We primarily followed the manufacturer’s protocols, however we
 extended the protocol of the Transketolase Activity Assay Kit to also
 measure latency. These modifications were based on published literature
 measuring transketolase latency (Engelhardt et al. 2020, Jones et al.
 2021). Frozen cod liver tissue (100 – 200 mg) was
 homogenized in a 2 ml cryovial with 1ml ice-cold Tris buffer and 2
 stainless steel balls (Steelball Lysing Matrix) for 1 min (6.5 ms^-^1;
 FastPrep-24 5G, MP Biomedicals). Samples were then centrifuged at 10,000 x
 g at 4°C for 15 mins. The supernatant (700 µl) was collected and filtered
 through a 10 kDA Spin Column. Protein concentrations (µg/ml) were measured
 according to the BCA Protein Assay Kit. To measure the transketolase
 activity and latency, lysates were diluted with Tris Buffer to reach
 protein concentrations ranging between 0.2-0.4 µg/µl. After mixing 30 µl
 lysate with 270 µl TKT Assay Buffer, 49 µl of the solution were placed in
 5 different wells on a 96-microtiter plate (Brand). We added 1 µl of 10 mM
 TDP (freshly solubilized in TKT Assay Buffer) to two wells to measure the
 stimulated transketolase activity. To the other three wells 1 µl TKT Assay
 Buffer was added to measure the basal transketolase activity in two wells
 and sample background activity in the last well. Reaction Mix (see
 manufacturer’s protocol, 50 µl) was added to wells prepared for the
 measurement of transketolase activity. Background Mix (50 µl) was added to
 sample background controls. Standards, substrate control, and positive
 control were prepared following the manufacturer’s protocol. We recommend
 adding a positive control for the latency measurement by including one
 sample of a specimen known to suffer from thiamine deficiency. This
 positive control should be prepared and run simultaneously with the actual
 samples. Using the microplate reader (FLUOstar Omega, BMG Labtech)
 preheated to 36°C, we measured the fluorescence (excitation: 544 nm;
 emission: 590 nm) every minute for 60 min. Following the manufacturer’s
 protocol, we calculated an average basal and stimulated transketolase
 activity for each specimen. The latency was calculated by dividing the
 basal activity by stimulated activity, subtracting it from 1 and
 multiplying it by 100. Wet and Dry Weights
 In cases where enough tissue remained after subsampling for the
 thiamine analysis, approximately 0.75 g of frozen tissue was weighed to an
 accuracy of 0.001 g before and after freeze-drying. Using the difference
 in weight, i.e., the water content, we estimate thiamine concentrations
 per wet or dry weight. Statistical analysis
 Statistical analyses were performed using R (version 4.4.2).
 Condition factor, used as a proxy for health, was estimated by extracting
 the residuals of a fitted model between somatic weight and length. Both
 variables were log-transformed. Individuals which could not be sexed due
 to immaturity were excluded from the analyses. To
 investigate how stock as well as several phenotypic variables may affect
 tissue-specific thiamine concentrations (per g wet weight), we constructed
 an initial linear mixed model (Gaussian distribution) including a
 three-way interaction between tissue type, maturity stage and sex along
 with two two-way interactions between tissue type and stock or length
 (used as a proxy for age) as well as condition factor as a fixed effect
 and the fish UID as a random effect (App. 1). After evaluating the
 three-way interaction, separate models were fitted for each tissue type
 (App. 2). These models included length, stock, condition factor, sex and
 maturity stage as fixed effects. An interaction effect between sex and
 maturity stage was tested for and kept in the model if significant. The
 model investigating thiamine concentrations in gonad tissue lacks sex as a
 fixed effect as it was only measured in female fish. In all models,
 thiamine concentrations were log-transformed and length was scaled. To
 control for non-independence among samples, models were fitted with fish
 UID as a random effect using the lme4 package. Model fit was evaluated
 using diagnostic plots of residual distribution with the DHARMa package.
 P-values were computed based on robust covariance matrix estimation, i.e.
 Wald tests. To control for potential effects of varying water content of
 the samples, we rerun all models using thiamine concentrations per dry
 weight (App. 1 & 2). To further investigate how
 the three stocks might be differing in their thiamine status and
 allocation, we calculated a total thiamine amount for somatic tissues per
 body weight. This was performed by calculating the total amount of
 thiamine in the liver tissue (liver weight multiplied by liver thiamine
 concentration) and other somatic tissues (eviscerated weight multiplied by
 muscle thiamine concentration used as an estimate) for each specimen. Both
 were combined and divided by the somatic weight. This provided an estimate
 of thiamine concentration per gram somatic weight. A model identical to
 the tissue-specific models (see above) was fitted. To
 study thiamine allocation between tissues throughout maturity, we
 calculated the total amount of thiamine in gonad, liver and muscle
 (eviscerated weight) by multiplying tissue weight with thiamine
 concentration for each specimen, i.e. their total thiamine pool. From
 there, the relative proportion of thiamine allocated to each tissue was
 estimated. Relative allocation of thiamine was plotted against maturity
 stages and analyzed visually. Similarly, the relative proportion of the
 three measured vitamers TF, TMP, and TDP were estimated and plotted
 against maturity stages. The effect of length, stock,
 condition factor and maturity stage on transketolase activity and latency
 was investigated by fitting models with aforementioned variables as fixed
 effects. References Bland B,
 Börjesson P. Expeditionsrapport IBTS, januari 2022. Institutionen för
 akvatiska resurser, Sveriges lantbruksuniversitet (SLU). eISBN:
 978-91-576-9972-5; 2022. Brown SB, Honeyfield DC,
 Vandenbyllaardt L. Thiamine Analysis in Fish Tissues. American Fisheries
 Society Symposium. 1998;21:73-81. Engelhardt J, Frisell
 O, Gustavsson H, Hansson T, Sjöberg R, Collier TK, et al. Severe thiamine
 deficiency in eastern Baltic cod (Gadus morhua). PLOS ONE.
 2020;15(1):e0227201. Heimbrand Y, Larsson S, Landfors
 F, Bergström U. Beståndsstatus för torsk i Ålands hav 2022. Institutionen
 för akvatiska resurser, Sveriges lantbruksuniversitet (SLU). SLU ID:
 SLU.Aqua. 2023.5.4-12; 2023. Jones KS, Parkington DA,
 Cox LJ, Koulman A. Erythrocyte transketolase activity coefficient (ETKAC)
 assay protocol for the assessment of thiamine status. Annals of the New
 York Academy of Sciences. 2021;1498(1):77-84. Lövgren
 O, Casini M. Expeditionsrapport BITS, februari/mars 2022. Institutionen
 för akvatiska resurser, Sveriges lantbruksuniversitet (SLU). eISBN:
 978-91-576-9973-2; 2022. Todisco V, Fridolfsson E, Axén
 C, Dahlgren E, Ejsmond MJ, Hauber MM, et al. Thiamin dynamics during the
 adult life cycle of Atlantic salmon (Salmo salar). Journal of Fish
 Biology. 2024;104(3):807-24. Tomkiewicz J, Tybjerg L,
 Holm N, Hansen A, Broberg C, Hansen E. Manual to determine gonadal
 maturity of Baltic cod. DFU rapport 116-02. Danish Institute for Fisheries
 Research. 2002.
description: # Thiamine allocation and deficiency status throughout the life cycle of
 cod Dataset DOI: [10.5061/dryad.cjsxksnjc](10.5061/dryad.cjsxksnjc) ##
 Description of the data and file structure We investigated the thiamine
 status of Atlantic cod of the North Sea, Baltic Proper and Åland Sea to
 better understand the prevalence of thiamine deficiency in these stocks
 and how thiamine is allocated throughout the life cycle of cod. Thiamine
 diphosphate, thiamine monophosphate, and free thiamine concentrations were
 measured in muscle, liver, and gonad tissues. The activity and latency of
 transketolase in liver was measured in a subset of specimens. For more
 details, see attached methods section or publication. ### Files and
 variables #### File: tbl_Fish.csv **Description:** Table containing
 general information about each specimen ##### Variables * fish_UID: (char)
 main UID for each fish * fish_UIDSVEA: (int) system-specific UID used in
 database on RV SVEA * fish_UIDOTOLITH: (int) otolith number UID used by
 SLU * fish_staUID: (int) UID for fishing hall * fish_staCRUISE: (char) UID
 for cruise (name/location + year + quarter) * fish_SEX: (char) sex (M/ F/
 empty = immature) * fish_WEIGHT: (num) total weight (g) * fish_LENGTH:
 (num) total length (mm) * fish_AGE: (int) age (counted from otoliths) *
 fish_LIVWEIGHT: (num) weight of liver (g) * fish_GONWEIGHT: (num) weight
 of gonad (g) * fish_GUTTEDWEIGHT: (num) gutted weight (g) *
 fish_INTWEIGHT: (num) weight of gastrointestinal tract (g) *
 fish_GONMATURITY: (int) category for gonad maturity (see method section)
 #### File: tbl_Thiamin.csv **Description:** Table containing thiamine
 concentrations ##### Variables * thia_fishUID: (char) main UID for each
 fish * thia_TISSUE: (char) tissue type * thia_DATE: (char) date of
 analysis * thia_VIAL: (int) vial number * thia_PREPPERS: (char) personal
 performing analysis * thia_TDP: (num) concentration of thiamine
 diphosphate (TDP; nmol/g) * thia_TMP: (num) concentration of thiamine
 monophosphate (TMP; nmol/g) * thia_TF: (num) concentration of free
 thiamine (TF; nmol/g) * thia_TTOT: otal thiamine (sum of TDP, TMP, and TF;
 nmol/g) #### File: tbl_Transketolase.csv **Description:** Table containing
 transketolase activity and latency ##### Variables * tk_RUN: UID of plate
 * tk_VIAL: Vial number * fish_UID: Main UID for each fish * tk_DILUTION:
 Dilution factor * tk_CONDITION: Category whether thiamine was added to the
 reaction (stimulated) or not (basal) * tk_TK: Transketolase activity
 (µUnits/µg protein) * tk_LATENCY: Transketolase latency (percent of
 unsaturated enzymes in system) * tk_TKAC: Transketolase activity
 coefficient (alternative calculation to latency) #### File:
 tbl_Watercontent.csv **Description:** Table containing information on
 water content of tissues ##### Variables * wc_fishUID: Main UID for each
 fish * wc_TISSUE: Tissue type * wc_WETWEIGHT: Wet weight of tissue (g) *
 wc_DRYWEIGHT: Dry weight of tissue (g) * wc_WCPERCENT: Water content of
 tissue (%) * wc_TCPERCENT: Tissue content (%) **Missing Data = NA** ##
 Code/software All statistical analyses were performed using R (version
 4.5.1). Session information can be found in the pdf file.