Are lipids always depleted? Comparison of hydrogen, carbon, and nitrogen isotopic values in the muscle and lipid of larval lampreys

Abstract

Stable isotope ratios in organisms can be used to estimate dietary source contributions, but lipids must first be accounted for to interpret values meaningfully. Lipids are depleted in heavy isotopes because during lipid synthesis light isotopes of carbon (12C) and hydrogen (1H) are preferentially incorporated. Prior work in larval lampreys has noted unusual lipid effects, which suggest lipids are enriched in the heavy isotope of carbon (13C), but still depleted in the heavy isotope of hydrogen (deuterium; 2H); nitrogen, a relatively rare element in lipids, has not been identified as being as sensitive to lipid content. Our objective was to determine if stable isotope ratios of hydrogen, carbon, and nitrogen behaved as expected in larval lampreys, or if their lipids presented different isotopic behavior. The δ2H, δ13C, and δ15N were measured from the muscle of four lamprey species before and after lipid extraction. In addition, muscle of least brook lamprey (Lampetra aepyptera) was collected every three months for a year from two streams in Maryland. Isotopic ratios were measured in bulk and lipid-extracted muscles, as well as in extracted lipids. The difference between muscle samples before and after lipid extraction (Δδ2H, Δδ13C, Δδ15N) was positively related to lipid proxy (%H or C:N ratio) and were fit best by linear models for Δδ2H and Δδ15N, and by a non-linear model for Δδ13C. The difference between lipid-extracted muscle and lipid δ13C (ΔMLδ13C) was negative and varied between months (ANOVA, F3,53 = 5.05, p < 0.005). Our work suggests that while lipids are often depleted in 13C, this is not a universal rule; however, the depletion of 2H in lipid synthesis appears broadly true.

Fig 1. Muscle sample a) δ²H, b) δ¹³C, and c) δ¹⁵N before and after lipid extraction.

Boxplot midlines are the median, the boxes enclose the 1^st-3^rd quartiles (i.e., 50% of the data points), and the whiskers are 1.5 times this interquartile range. Measured values that boxplots are drawn from are represented as points and are jittered to make them more easily distinguishable. The difference before and after extraction for each paired value is shown as a light grey line between groups.

Fig 2

a) The δ²H_bulk of muscle and the C:N ratio measured during δ¹³C/δ¹⁵N analysis in a separate subsample. b) The percent hydrogen in a δ²H_bulk sample regressed against the C:N ratio measured during δ¹³C/δ¹⁵N analysis in another subsample. c) The muscle δ²H_bulk sample regressed against the percent hydrogen which is measured simultaneously to δ²H. Also shown on plots are the linear fits (solid black lines), the 95% confidence intervals of the fit (grey region), the linear model’s equation, and its R² values. Although not used for linear fitting, the samples are colored by month of collection, and the species collected is denoted by shape (ABL = American brook lamprey, LBL = least brook lamprey, SL = sea lamprey).

Fig 3

a) The δ¹³C_bulk and b) the δ¹⁵N_bulk of lamprey muscle and their C:N ratio, colored by the month in which they were collected. The species collected is denoted by shape (ABL = American brook lamprey, LBL = least brook lamprey, SL = sea lamprey).

Fig 4

a) The Δδ²H (δ²H_extracted−δ²H_bulk) of muscle samples regressed against the percent hydrogen in the unextracted muscle sample. b) The Δδ¹³C (δ¹³C_extracted−δ¹³C_bulk) and the c) Δδ¹⁵N (δ¹⁵N_extracted−δ¹⁵N_bulk) of muscle samples regressed against the C:N ratio in the bulk muscle sample. The lines of best fit are overlain; the solid line is y = ax +b, the dashed line is y = a∙ln(x) + b, the dotted line is y = (ax + b)/(x + c), and the dot-dash line is y = (ax + b)/x.

Fig 5

a) The Δ_MLδ²H (δ²H_extracted−δ²H_lipid) and b) the Δ_MLδ¹³C (δ¹³C_extracted−δ¹³C_lipid) plotted by the month of collection and color for the river they were collected from.