Abstract
Macronutrients, comprising carbohydrates, proteins and lipids, underpin many ecological processes, but their quantification in ecological studies is often inaccurate and laborious, requiring large investments of time and bulk samples, which make individual‐level studies impossible. This study presents Macronutrient Extraction and Determination from Invertebrates (MEDI), a protocol for the direct, rapid and relatively low‐cost determination of macronutrient content from single small macroinvertebrates.Macronutrients were extracted by a sequential process of soaking in 1:12 chloroform:methanol solution to remove lipid and then solubilising tissue in 0.1 M NaOH. Proteins, carbohydrates and lipids were determined by colorimetric assays from the same individual specimens.The limits of detection of MEDI with the equipment and conditions used were 0.067, 0.065 and 0.006 mg/ml for proteins, carbohydrates and lipids respectively. Adjusting the volume of reagents used for extraction and determination can broaden the range of concentrations that can be detected. MEDI successfully identified taxonomic differences in macronutrient content between five insect species.Macronutrient Extraction and Determination from Invertebrates can directly and rapidly determine macronutrient content in tiny (dry mass ~3 mg) and much larger individual invertebrates. Using MEDI, the total macronutrient content of over 50 macroinvertebrates can be determined within around 3 days of collection at a cost of ~$1.35 per sample.
Highlights
Carbohydrates and lipids were determined by colorimetric assays from the same individual specimens
Samples were first weighed and lipids were extracted by soaking whole arthropods in 1 ml of 1:12 chloroform:methanol for 24 hr
This procedure for soaking arthropods was repeated for another 24 hr, but discarding all supernatant, to ensure any residual lipids were removed from the sample prior to protein and carbohydrate extraction
Summary
Despite the relevance of macronutrients, comprising proteins, carbohydrates and lipids, to a broad range of applications, few ecological studies quantify them. Using MEDI, the total carbohydrate, lipid and protein content of over 50 macroinvertebrates can be determined within around 3 days of collection at a cost of ~$1.35 per sample using standard laboratory equipment. This protocol will enhance the study of macronutrient content in invertebrates and other small samples in contexts including trophic interactions, parasitology and development. Macronutrient Extraction and Determination from Invertebrates successfully determined protein, carbohydrate and lipid content directly in parallel from a range of invertebrates, with a turnaround time from sample to data of 3 days and at a cost of ~$1.35 per sample using standard laboratory equipment (heating block, shaker, bead beater and plate reader). The gravimetric lipid mass and exoskeletal mass were determined for the three focal species for which body mass could be accurately measured (Table 4)
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