title: The effect of lipid extraction on carbon and nitrogen stable isotope ratios in oyster tissues: Implications for glycogen-rich species.
abstract: BACKGROUND
  Extraction of lipids from tissues prior to carbon stable isotope analysis (SIA) has become a common practice, despite a lack of species-specific data to indicate when lipid extraction is needed. Marine invertebrates, including bivalves, are known to store carbon as glycogen and less in the form of lipids than other species, potentially reducing the need for lipid extraction even when C:N values are above 3.5, a value that previous studies suggest indicates a need for lipid extraction of animal tissues.
  
  
  METHODS
  We investigated the need for lipid extraction on individual tissues (adductor muscle, gut gland, gill) and whole tissue of a glycogen-storing species, the oyster Crassostrea virginica. Bulk and lipid-extracted samples were analyzed for their C and N stable isotope ratios by continuous flow isotope ratio mass spectrometry (IRMS). Samples were analyzed on a 20-20 isotope ratio mass spectrometer (PDZ Europa) after combustion in an elemental analyzer (PDZ Europa Automatic Analyzer-Gas Solid Liquid).
  
  
  RESULTS
  Although the C:N values for most bulk (unextracted) tissue samples were greater than 3.5, the lipid-extracted δ13C values did not differ from the bulk values. Lipid extraction, however, affected δ15N values in all tissue types except adductor muscle, indicating that separate SIA may be required when tissues are lipid extracted.
  
  
  CONCLUSIONS
  These data demonstrate that it is not necessary to lipid extract oyster tissues in all cases, and that C:N thresholds for lipid extraction in other species may not be reliable for organisms such as oysters that store glycogen. Our data indicate that minimizing unnecessary lipid extraction through preliminary testing will save researchers time and expense by avoiding superfluous sample handling, reducing concern over secondary effects on data quality, and reducing the costs of reagents and additional separate stable isotope analysis to ensure analytical accuracy. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Department of Marine Sciences, University of South Alabama, Mobile, AL, 36688, USA.|Dauphin Island Sea Lab, Dauphin Island, AL, 36528, USA.|Department of Biology, University of Alabama Birmingham, Birmingham, AL, 35294, USA.
authors.forename: H K
authors.initials: HK
authors.lastname: Patterson
authors.affiliation: Department of Marine Sciences, University of South Alabama, Mobile, AL, 36688, USA.|Dauphin Island Sea Lab, Dauphin Island, AL, 36528, USA.
authors.forename: R H
authors.initials: RH
authors.lastname: Carmichael
mesh_terms: D000818:Animals; D002247:Carbon Isotopes / Q000032:analysis*; D006003:Glycogen / Q000032:analysis*; D008055:Lipids / Q000737:chemistry* / Q000302:isolation & purification; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes / Q000032:analysis*; D010125:Ostreidae / Q000737:chemistry*; D012758:Shellfish / Q000032:analysis*
chemical_list: D002247:Carbon Isotopes; D008055:Lipids; D009587:Nitrogen Isotopes; D006003:Glycogen