Arithmetic normalization models for the effects of lipid on carbon stable isotope values in silver carp (Hypophthalmichthys molitrix) tissue

Invasive silver carp (Hypophthalmichthys molitrix) threaten Mississippi River basin ecosystems due to their ability to outcompete native species. Stable carbon (δ13C) and nitrogen (δ15N) isotope analysis has been used in prior studies to help identify how silver carp impact native ecosystems, however lipids in fish tissues commonly bias their δ13C values. Chemical lipid extraction and mathematical equations that normalize δ13C values for lipid content are common ways to account for the effect of lipid on tissue stable isotope values in fish, though species and tissue-specific arithmetic models have not yet been derived for silver carp. Using paired t-tests, we assessed differences in δ13C, δ15N, and C:N ratios before and after chemical lipid extraction in muscle and whole fish from silver carp collected along three sites in the Mississippi River. We then used linear and natural log models to estimate lipid-extracted δ13C values in carp muscle and whole fish samples based on their non-lipid-extracted δ13C values and elemental C:N ratios. We evaluated these arithmetic models for best fit and parsimony and used paired t-tests to assess accuracy between mathematically normalized and chemically lipid-extracted δ13C values. We found that chemical lipid extraction increased silver carp δ13C values and decreased C:N ratio in muscle and whole fish as well as increasing δ15N values in whole fish but not fish muscle. While both linear and natural log regression models accurately estimated lipid extraction δ13C in both sample types, the natural log approach provided better model fits and greater parsimony throughout the wide range of C:N ratios observed in silver carp. Our results confirmed the need to account for lipid-effects on δ13C values in silver carp tissue. Moreover, the arithmetic models provided by our study will allow future researchers to conduct stable isotope analysis on silver carp without the added time and cost of chemical lipid-extraction. In addition, the positive relationship we identified between fish muscle and whole fish will facilitate the comparison of stable isotope values in silver carp across studies.

Methods
Thirty-two silver carp from three regions (one in Illinois and two in Louisiana) along the Mississippi River watershed were collected in 2023 and 2024. Each fish was thawed, weighed, and measured and aliquots of white muscle and whole ground fish were sampled, freeze-dried and split in half to create a replicate for lipid extraction. Lipids were chemically extracted using Soxhlet extraction overnight (22-24 hours) in a 2:1 chloroform-methanol solvent and air-dried upon removal. Dry weights were taken before and after lipid extraction to calculate the percentage of lipid as a function of each samples dry weight. Whole fish and fish muscle samples were analyzed before (NLE) and after lipid extraction (LE) for carbon (C) and nitrogen (N) elemental concentration (%C and %N) and ratios (C:N) as well as stable isotope values (δ13C, δ15N) using an elemental analyzer system coupled to a continuous-flow stable isotope ratio mass spectrometer.