DOI: 10.5061/dryad.47d7wm3g3
title: Data for effects of MeHg on avian lipid metabolism

description: Methylmercury (MeHg) is a global pollutant that can cause metabolic
 disruptions in animals and thereby potentially compromise the energetic
 capacity of birds for long-distance migration, but its effects on avian
 lipid metabolism pathways that support endurance flight and stopover
 refueling have never been studied. We tested the effects of short-term
 (14-d), environmentally relevant (0.5 ppm) MeHg exposure on multiple lipid
 metabolism markers in the pectoralis and livers of yellow-rumped warblers
 (Setophaga coronata) that were previously found to have poorer flight
 endurance in a wind tunnel than untreated conspecifics. Compared to
 controls, MeHg-exposed birds displayed lower muscle aerobic and fatty acid
 oxidation capacity, but similar muscle glycolytic capacity, fatty acid
 transporter expression, and PPAR expression. Livers of dosed birds
 indicated elevated energy costs, lower fatty acid uptake capacity, and
 lower PPAR-γ expression. The lower muscle oxidative enzyme capacity of
 dosed birds may have caused or contributed to their weaker endurance in
 the prior study, while the metabolic changes observed in the liver have
 potential to inhibit lipogenesis and stopover refueling. Our findings
 provide concerning evidence that fatty acid catabolism, synthesis, and
 storage pathways in birds can be dysregulated by only brief exposure to
 MeHg, with potentially significant consequences for migratory performance.
description: We analyzed tissues that were collected from 24 yellow-rumped
 warblers used by Ma et al. [17] to study the effects of MeHg on flight
 performance in a wind tunnel. The reader is directed to Ma et al. [17] for
 detailed methods regarding capture, husbandry, dosing, and wind tunnel
 testing. Briefly, the birds were captured during migratory stopovers at
 Long Point, Ontario, Canada in the autumn of 2014 and transported to
 indoor aviaries at the University of Western Ontario in London, Canada.
 There they were maintained on an <em>ad libitum</em>
 mercury-free, synthetic diet under light cycles that simulated autumn
 (12L:12D) and then winter (9L:15D) photoperiods until March of 2015, when
 the photoperiod was lengthened (16L:8D) to stimulate the birds into spring
 migratory condition. After 14 d of the lengthened photoperiod, 12 randomly
 selected birds (4 male, 8 female) were switched to an <em>ad
 libitum</em> synthetic diet containing 0.5 ppm ww MeHg for 14 d
 while the other 12 birds (3 male, 9 female) continued to feed on the same
 mercury-free diet to serve as a control group. The dosing concentration of
 0.5 ppm ww was intended to represent the middle to upper range of MeHg
 levels commonly found in the arthropod prey of songbirds in eastern North
 America [4,27,28]. Blood samples were collected by
 brachial venipuncture on days 0 and 14 of the experiment, and stored at
 -80º C. Total body mass was measured to 0.01 g on a digital balance, and
 fat mass and lean mass were measured to 0.001 g with quantitative magnetic
 resonance analysis, also on days 0 and 14. On day 14, 22 of the 24 birds
 were flown for up to 2 h in a wind tunnel, after which they were
 immediately euthanized. Two individuals that could not be flown in the
 wind tunnel because of missing tail feathers were also euthanized on day
 14 for inclusion in the present study. Pectoralis muscles and livers were
 then collected from all 24 birds immediately following euthanasia and
 stored at -80º C. Dosed and control birds did not differ in body size
 (wing length) upon capture, or body mass or body composition at the start
 or end of the 14-d experiment [17].
 <strong>Laboratory analyses</strong>
 <em>Tissue mercury concentrations</em>
 We measured blood total mercury (THg; ww) as a proxy for MeHg
 using a direct mercury analyzer (DMA-80, Milestone Inc., Shelton, USA), as
 described in Ma et al. [17]. The instrument was calibrated with a
 certified reference material (Caprine Blood SRM 955c) and quality control
 was assessed with a method blank, certified concentration standard (CCS),
 and sample duplicates. Mean percent recoveries were 104.33 ± 3.37% (SRM
 955c; n = 8), 92.15 ± 0.98% (DORM-2; N = 3), and 97.85 ± 0.81% (CCS; N =
 36). CVs of duplicates averaged 6.84% ± 1.40%. We estimated pectoralis and
 liver THg concentrations based on blood THg concentrations, using
 regression equations from Ma et al. [17].
 <em>Metabolic markers</em> We
 measured citrate synthase (CS) and carnitine palmitoyltransferase (CPT)
 activity to assess aerobic capacity and fatty acid oxidation capacity,
 respectively [29-31]. We measured the gene expression of fatty acid
 transport proteins to assess muscular and hepatic fatty acid uptake
 capacity. They included fatty acid translocase (FAT/CD36) and plasma
 membrane fatty acid binding protein (FABPpm), to evaluate membrane fatty
 acid uptake capacity, and heart-type fatty acid binding protein (H-FABP),
 to evaluate intracellular fatty acid transport capacity (pectoralis only)
 [20,21,30]. We then measured expression of peroxisome
 proliferator-activated receptors (PPAR-α,
 PPAR-ß, PPAR-γ),
 which are nuclear receptors that regulate lipid metabolism in birds [32].
 PPAR-α and PPAR-β are highly expressed in heart and skeletal muscle where
 they mainly target genes for proteins involved in lipid oxidation,
 including CPT and FAT/CD36 [33,34]. PPAR-γ, in contrast, primarily
 controls expression of FABPpm, FAT/CD36, and lipogenic enzymes in the
 liver and adipocytes to regulate lipid synthesis and storage [33,34].
 Lastly, we measured lactate dehydrogenase (LDH) activity to assess effects
 of MeHg on glycolytic capacity given the broad ability of MeHg to
 inactivate enzymes [35] and the effect this could have on migrating birds
 early in flight, before they switch over from catabolizing primarily
 carbohydrates to primarily fatty acids [36].
 <em>Enzyme assays</em> We homogenized 50-100 mg of pectoralis and liver
 using beadmill homogenization (NextAdvance, Troy, NY; speed 8, time 3 for
 liver and time 4 for pectoralis, 4°C) in 9 volumes of 20 mM Na2PO4, 0.5 mM
 EDTA, 0.2% BSA, 0.1% Triton x-100, and 50% Glycerol, pH = 7.4. We then
 assayed CS, CPT, and LDH in duplicate or triplicate in a microplate reader
 (BioTek, Winooski, VT) at 39°C to represent a typical avian body
 temperature. We measured LDH at 340 nm using 0.4 mM pyruvate, 0.66 mM
 NADH, 5 mM DTT, 50 mM Tris, pH = 7.4. For CS and CPT, we measured
 absorbance at 412 nm to detect the appearance of CoA-TNB as in Price et
 al. [37]. <em>qPCR of transport
 proteins and PPARs</em> We homogenized ~50 mg of pectoralis and liver in 1
 mL of TriZol (Invitrogen, Carlsbad, CA) and separated RNA into an aqueous
 phase using a chloroform ethanol procedure. We purified total RNA using a
 PureLink RNA kit (Invitrogen, Carlsbad, CA) and quantified RNA using a
 plate adapter (BioTek, Winooski, VT). We treated 14 µg
 of RNA using TURBO I DNase (Invitrogen,
 Carlsbad, CA) and used Nanodrop 2000 (Thermo Fisher Scientific, Waltham,
 MA) to quantify and check quality before reverse transcription of
 1 μg of DNase-treated RNA using NEB Luna RT (New England Biolabs, Ipswich,
 MA). We
 selected Glyceraldehyde 3-phosphate dehydrogenase
 (<em>GAPDH</em>) as a housekeeping gene for qPCR using a
 published primer sequence for yellow-rumped warblers [38] and used
 published primer sequences for FAT/CD36, FABPpm,
 H-FABP, and PPARs
 [39-41]. Each cDNA sample was
 diluted 1:5 with nuclease-free water before analysis. We confirmed primer
 specificity on a 2% agarose gel and used a serial dilution series of
 pooled cDNA to obtain amplification efficiency (between 97.1% and 99.9%).
 Reactions were run in duplicate in a StepOnePlus qPCR machine (Applied
 Biosystems, Foster City, CA; 60s at 95°C, followed by 40 cycles at 95°C
 for 15s and 60°C for 30s) in 10 µL volumes with 0.2 µM primers and 1 µL
 cDNA in Luna Universal qPCR master mix (New England Biolabs, Ipswich, MA).
 We used comparative cycle threshold (C<sub>T</sub>) analysis
 following Schmittgen and Livak [42], subtracting the average
 C<sub>T</sub> for the corresponding housekeeping gene reading
 (<em>GAPDH</em>) from the average C<sub>T</sub>
 from duplicate target gene wells to obtain the ΔC<sub>T</sub>
 for each individual and gene, then subtracting the control group mean ΔCT
 from the MeHg-dosed group mean to obtain ΔΔC<sub>T</sub>. Fold
 change was calculated for the mean and upper and lower limits of the SEM
 using 2-<sup>(ΔΔCT)</sup>.
 <strong>Statistical
 analyses</strong> We compared blood
 THg concentrations between treatment groups with a two-tailed t-test. We
 tested the effects of treatment on each enzyme, transport protein, and
 PPAR isoform using general linear models. We included lean mass, fat mass,
 the lean mass by treatment interaction, and fat mass by treatment
 interaction in full models which we then compared to nested models with
 likelihood ratio tests to determine whether any of these terms could be
 dropped. We retained a term for inclusion in a final model along with
 treatment when a likelihood ratio test of the full model against the
 nested model from which it was removed had a P value of &lt; 0.1. We
 used lean mass also as a proxy for body
 size (wing length) and sex, as all three variables were significantly
 related to each other. We performed all statistical analyses in R (v
 4.0.4, R Foundation for Statistical Computing, Vienna, Austria) and
 accepted significance when P &lt; 0.05. We log-transformed non-normal
 variables prior to analyses and removed outliers that were likely
 measurement errors, such as unrealistically high or low values, an error
 in the housekeeping gene, or a consistent error for a set of tissue
 samples run together.