Triacylglyceride measurement in small quantities of homogenised insect tissue: Comparisons and caveats Caroline M. Williams a,⇑ , Raymond H. Thomas b , Heath A. MacMillan a , Katie E. Marshall a , Brent J. Sinclair a a Department of Biology, University of Western Ontario, London, Ontario, Canada b Department of Psychology, University of Western Ontario, Canada article info Article history: Received 9 June 2011 Received in revised form 4 August 2011 Accepted 5 August 2011 Available online 22 August 2011 Keywords: Storage lipid Triacylglyceride Sulfo-phospho-vanillin Enzymatic Gravimetric TLC-FID abstract Triacylglycerides (TAGs) are the most important stored energy reserve in eukaryotes and are regularly measured in insects. Quantitative analysis of TAGs is complicated by their diversity of structure, and there are concerns with the quantitative accuracy of commonly used analytical methods. We used thin layer chromatography coupled to a flame ionisation detector (TLC-FID), an accurate method that is not sensitive to saturation or chain length of fatty acids, to quantify TAG content in small amounts of insect tissue, and used it to validate three high-throughput lipid assays (gravimetric, vanillin, and enzymatic). The performance of gravimetric assays depended on the solvent used. Folch reagent (chloroform: meth- anol 2:1 v/v) was a good index of TAG content, but overestimated lipid content due to the extraction of structural lipid and non-lipid components. Diethyl ether produced reasonable quantitative measure- ments but lacked precision and could not produce a repeatable rank-order of samples. The vanillin assay was accurate both as a quantitative method and as an index, preferably with a standard of mixed fatty acid composition. The enzymatic assay did not accurately or precisely quantify TAGs under our assay con- ditions. We conclude that the vanillin assay is suitable as a high-throughput method for quantifying TAG providing fatty acid composition does not change among treatment groups. However, if samples contain significant quantities of di- or mono-acylglycerides, or the fatty acid composition differs across treatment groups, TLC-FID is recommended. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Storage lipids, or triacylglycerides (TAGs), are the dominant energy reserve in eukaryotic organisms (Bell and Coleman, 1980), and the storage, mobilization, and metabolism of fat stores in insects are key metabolic processes (Fast, 1964; Gilby, 1965; Fast, 1970; Downer and Matthews, 1976; Beenakkers et al., 1985; Arrese and Soulages, 2010). Insect lipid stores are key fitness deter- minants in overwintering insects, especially those which rely on lipid catabolism to meet metabolic demands over winter and to fuel metamorphosis and reproduction in the spring (Hahn and Denlinger, 2011), and are widely used as indirect measures of in- sect performance and fitness (e.g. Otronen, 1995; Ojeda-Avila et al., 2003; Hahn, 2005; Huho et al., 2007; Bosch et al., 2010). Triacylglycerides are comprised of a trihydric alcohol glycerol esterified with three long chain fatty acids (acyl chains). Hydrocar- bon chain length, number and position of double bonds, and rela- tive molecular mass of the acyl chains will all influence the polarity and chemical reactivity of TAG molecules (Nikolova-Damyanova, 1999), which can lead to systematic biases in some methods of quantification. The presence of other neutral lipids with similar chemical properties but different functional significance (e.g. mono- and di-acylglycerides) makes the development of a specific assay difficult, particularly when multiple tissue types are present in a sample. Small body size and a diffuse fat storage organ (the fat body) (Downer and Matthews, 1976; Arrese and Soulages, 2010) also complicate lipid analysis in insects and make techniques such as condition indices or fat scoring (Krementz and Pendelton, 1990), magnetic resonance imaging (McGuire and Guglielmo, 2010), or dual energy x-ray absorptiometry (Stevenson and van Tets, 2008) less accessible than they are to researchers working on larger 0022-1910/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2011.08.008 Abbreviations: BHT, butylated hydroxytoluene; CE, cholesterol ester; DAG, diacylglycerol; DM, dry mass; FFA, free fatty acid; FM, fresh mass; GC, gas chromatography; GLY, glycerol; HPLC, high-pressure liquid chromatography; LFDM, lipid-free dry mass; MAG, monoacylglycerol; TAG, triacylglycerol; TLC-FID, thin layer chromatography coupled to a flame ionisation detector; TLL, trilinolein; TOL, triolein; TPT, tripalmitin. ⇑ Corresponding author. Address: Biological and Geological Sciences Building, Room 2056, Department of Biology, The University of Western Ontario, London, Canada ON N6A 5B7. Tel.: +1 519 661 2111x89158; fax: +1 519 661 3935. E-mail address: cwilli67@uwo.ca (C.M. Williams). Journal of Insect Physiology 57 (2011) 1602–1613 Contents lists available at SciVerse ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys