Blood Plasma Volume

DNA level in diluted samples was measured by SYBR Green dye-based qPCR assay using a PRISM 7300 sequence detection system (Applied Biosystems). The primer sequences were as follows: human NADH dehydrogenase 1 gene (mtDNA): forward 5′-ATACCCATGGCCAACCTCCT-3′, reverse 5′-GGGCCTTTGCGTAGTTGTAT-3′[7] (link),[24] (link); human β-globin (nuclear DNA): forward 5′-GTGCACCTGACTCCTGAGGAGA-3′, reverse 5′-CCTTGATACCAACCTGCCCAG-3′[25] (link); bacterial 16S ribosomal RNA: forward 5′- CGTCAGCTCGTGTTGTGAAA-3′, reverse 5′-GGCAGTCTCCTTGAGTTCC-3′[7] (link). Plasmid DNA with complementary DNA sequences for human mtDNA was obtained from ORIGENE (SC101172), and plasmid DNA with complementary DNA sequences for human nuclear DNA was obtained from Sino Biological. Concentrations were converted to copy number using the formula; mol/gram×molecules/mol = molecules/gram, via a DNA copy number calculator (http://cels.uri.edu/gsc/cndna.html; University of Rhode Island Genomics and Sequencing Center) [26] (link),[27] (link). DNA solutions were diluted in 10-fold serial dilutions and used as standards.
The thermal profile for detecting mtDNA was carried out as follows: an initiation step for 2 min at 50°C is followed by a first denaturation step for 10 min at 95°C and a further step consisting of 40 cycles for 15 s at 95°C and for 1 min at 60°C. A representative standard curve, dissociation curve, and amplification plot are shown in Figure 1.
All samples were analyzed in duplicate, and a no-template control was included in every analysis. mtDNA levels in all of the plasma analyses were expressed in copies per microliter of plasma based on the following calculation [28] (link): where c is the concentration of DNA in plasma (copies/microliter plasma); Q is the quantity (copies) of DNA determined by the sequence detector in a PCR; V_DNA is the total volume of plasma DNA solution obtained after extraction, typically 200 µl per extraction; V_PCR is the volume of plasma DNA solution used for PCR, typically 5 µl of ten times diluted plasma DNA solution; and V_ext is the volume of plasma extracted, typically 50–100 µl.
The specificity of mtDNA primers was examined using bacterial DNA (Text S2 in Supporting Information S1; Figure S1) and by BLAST search (Text S3 and Tables S1 and S2 in Supporting Information S1).

Human whole blood was collected and supplied by the Volunteer Blood Donor Registry (Clinical Translation Centre, Walter & Eliza Hall Institute of Medical Research, Parkville, Australia) and used on the day of collection. The haematocrit (Hct) was determined by centrifugation (13,000×g for 3 min using Clemets^® Microhaematocrit centrifuge and Safecap^® Plain Self-sealing Mylar wrapped capillary tubes) to ensure it was between 0.40 and 0.48. An aliquot was centrifuged (Heraeus, Multifuge 3 S-R; 4500×g) for 10 min to obtain plasma required for matrix matching purposes as described below.
Aliquots of whole blood were spiked with compound stock solutions (prepared in 20/40/40 (v/v) DMSO/acetonitrile/water) to give a final nominal concentration of 1000 ng/mL with final DMSO and acetonitrile concentrations of 0.2% (v/v) and 0.4% (v/v), respectively. Two aliquots of the spiked whole blood were transferred to fresh microcentrifuge tubes and maintained at 37 °C/5% CO₂ in a humidified incubator. The pH was confirmed to be 7.4 ± 0.1 at the start and end of the incubation. At each time point (30 min and 4 h), one whole blood tube was removed from the incubator and mixed by gentle inversion, after which four replicate blood samples were taken and matrix matched with an equal volume of blank plasma. The remainder of the blood sample was centrifuged (Eppendorf, Mini Spin plus; 6700×g) for 2 min for the collection of 4 replicate plasma samples which were similarly matrix matched with an equal volume of blank whole blood. The 1:1 mixtures of blood/plasma were mixed, snap frozen in dry ice and stored at − 80 °C until analysis by LC–MS (Additional file 1: Table S2) against calibration standards prepared in the same mixed matrix. Any further distribution of compound into RBCs at this stage was irrelevant as the cells were lysed during the sample preparation and the total concentration in the mixed matrix was measured for both the calibration standards and samples.
Compound stability in whole blood was assessed by comparing the compound concentrations measured at 30 and 240 min. The apparent whole blood-to-plasma partitioning ratio (B/P) was calculated as the ratio of the average concentration in the blood sample to that in the plasma fraction of the same whole blood sample. A standard deviation (SD) for each B/P value was calculated using the propagation of errors approach as described previously [38 (link)].

To model our observed reproductive processes from Firkus et al. (2022) (link) in our lake trout DEB, we added an egg module that allows reproductive hormone dynamics to dictate the conversion of energy in the reproductive buffer into eggs. Previous laboratory studies suggest that estradiol concentration modulates the effects of sea lamprey parasitism on lake trout reproduction (Smith et al., 2016 (link); Firkus et al., 2022 (link)), so it is necessary to account for estradiol's role in our model. A similar approach to incorporating hormone dynamics into a DEB model is outlined in Murphy et al. (2018) (link) and Muller et al. (2019) (link); however, we simplified this approach so that estradiol concentration was the only required input. This approach more explicitly describes the processes involved in egg development and allowed the model to account for differences in estradiol concentration in parasitized and unparasitized individuals. In the egg module, reproductive reserve (energy available for use towards reproduction) molecules are combined with estradiol to synthesize the egg yolk protein vitellogenin. Processes that take place in the blood plasma volume or liver are taken proportional to the structural mass. The energy flux for egg mass production is triggered by estradiol density of (i.e. the ratio of mass of estradiol in plasma and the structural mass, and follows the law of mass action with the reproductive reserve density ( ). Vitellogenin production occurs in the liver and is secreted into plasma and travels to the ovaries where it is absorbed by ovarian follicles; all processes involved are proportional to the structural mass of the fish . Thus, the rate of egg mass production is given by , where the parameters describing the conversion of reserve, estradiol and vitellogenin to egg mass are absorbed in the proportionality constant . The dynamics of the egg ovarian mass, , are given in Table 1.
Data for estradiol were obtained from laboratory studies of siscowet lake trout (Firkus et al., 2022 (link)) as ng/ml of plasma. These data were linked to the model variable that accounts for the mass of estradiol (in C-mol): , where is the estradiol concentration (ng/ml of plasma), is the molecular weight of estradiol (15.1 g/C-mol) and is the total volume of plasma in a lake trout in ml given by the following equation: , where is wet weight and is the proportionality constant (averaged value of 2.86% from Gingerich et al., 1987 (link) and Gingerich and Pityer, 1989 (link)). The total wet weight has contributions from structural mass, reserve mass and ripe ( and unripe ( r reproductive mass: , where and are the molecular weights and densities of structure and reserve, respectively (Table 3).

The HemoCue system (Glucose 201+, Hb201, and WBC DIFF, HemoCue AB, Angelholm, Sweden) was used to determine blood glucose concentration, haemoglobin, total and differential leukocyte counts (including neutrophils, lymphocytes and monocytes) in duplicate from whole blood samples. The coefficient of variation (CV) for blood glucose concentration, haemoglobin and leukocyte counts was 5.1%, 1.6%, and 13.6% respectively. Haematocrit was determined by capillary method in triplicate from heparin whole blood samples using a microhematocrit reader (CV: 0.7%) (Thermo Fisher Scientific). The haemoglobin and haematocrit values were used to determine changes in plasma volume (P_v) relative to baseline, and to correct plasma variables (Dill and Costill, 1974 (link)). Bacterially-stimulated elastase release was determined as previously described (Costa et al., 2009 (link); Costa et al., 2011 (link); Costa et al., 2019a (link); Costa R. J. S. et al., 2020 (link)). The remaining whole blood in the heparin and K3EDTA vacutainers was centrifuged at 4,000 rpm (1,500 g) for 10 min within 15 min of sample collection and aspirated into 1.5 ml micro-storage tubes and frozen at −80°C until analysis. Prior to freezing, two 50 µl aliquots of heparin plasma were used to determine plasma osmolality (P_Osmol), in duplicate (CV: 0.7%), by freeze point osmometry (Osmomat 030, Gonotec, Berlin, Germany). Circulating concentrations of cortisol (DKO001; DiaMetra, Italy), PMN elastase (BMS269; Affymetrix EBioscience, Vienna, Austria), I-FABP (HK406; Hycult Biotech, Uden, Netherlands), sCD14 (HK320; Hycult Biotech), and LBP (HK315, Hycult Biotech) were determined by ELISA. Additionally, systemic cytokine profile [i.e., plasma IL-1β, TNF-α, IL-10, and IL-1ra concentrations] (HCYTMAG-60K, EMD Millipore, Darmstadt, Germany) were determined by multiplex system (Magpix, Luminex, Austin, TX, United States). All variables were analysed as per manufacturer’s instructions on the same day, with standards and controls on each plate, and sample from each participant assayed on the same plate. The intra- and inter-assay CV for analysed biomarkers, respectively, was 6.1% and 10.4% for cortisol, 2.8% and 3.6% for I-FABP, 4.0% and 9.3% for LBP, 3.3% and 4.2% for sCD14, 5.5% and 9.7% for elastase, 16.0% and 16.6% for IL-1β, 14.9% and 15.5% for TNFα, 15.8% and 9.1% for IL-6, 14.7% and 12.6% for IL-8, 15.9% and 11.1% for IL-10, and 9.2% and 8.8% for IL-1ra.