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 MEDI (Macronutrient Extraction and Determination from Invertebrates), 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 solubilizing 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 mg ml-1, 0.065 mg ml-1 and 0.006 mg ml-1 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.

MEDI 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 three days of collection at a cost of ~$1.35 per sample.

Description and Implementation

Materials

All materials, unless stated otherwise, were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Flat bottom, 96-well microplates (Sterilin Microplate F Well), Pierce BCA Protein Assay reagents and Pierce Modified Lowry Protein Assay reagents were obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Ribbed, skirted 1.5 ml screwcap microtubes and caps were obtained from STARLAB (Hamburg, Germany). Sulfuric acid (95%) and phosphoric acid (85%) were obtained from Fisher Scientific (Pittsburgh, Pennsylvania, USA).

Macronutrient extraction

Macronutrient extraction is a two-step process that first involves extracting lipid and then solubilizing the remaining tissue for carbohydrate and protein analysis (Figure 1-2). Details of the methods will vary depending on the size of arthropod used. There are many important considerations when analysing the macronutrient content of arthropods (Table 1).

 

Figure 1: Workflow of MEDI for specimens of different sizes.

Figure 2: Protocol for the extraction of macronutrients and measurement of exoskeletal mass from invertebrate bodies. Only the first two rows are carried out for macronutrient extraction. For exoskeletal measurement, separate samples were used in this study. It is advised to lyse the specimen for protein extraction because this allows efficient extraction of protein in one wash of NaOH. But it is not advised to lyse the specimen for exoskeletal measurement because preliminary work suggests that exoskeleton measurements on lysed samples are significantly lower than measurements on intact samples (Wilder unpublished); it is also advised to heat the specimen for a longer period (i.e. 2 h) and to repeat the central steps (addition of NaOH, heating, incubation and discarding of supernatant) to ensure removal of all soft tissues. Figure created using Biorender.com.

Table 1: Considerations when analyzing the macronutrient content of arthropod samples.

Technique	Options	Information	Best Practice Suggestion
Protein Assay	Crude Protein (6.25 x % nitrogen)	Assumes that all nitrogen in a sample is in the form of protein with 16% nitrogen.	The estimated protein content of a sample will vary depending on the method used and each has biases. Ideally, analysis of hydrolyzed amino acids could be used to determine which assay is most appropriate for a group of organisms. Alternatively, users can measure samples using multiple assays and take the average of those estimates.
	Bradford	Primarily reacts with arginine, lysine, and histidine
	BCA	Primarily reacts with cysteine/cystine, tyrosine, and tryptophan
	Lowry	Primarily reacts with cysteine/cystine, tyrosine, and tryptophan
	Hydrolyzed Amino Acid Analysis	Considered one of the most accurate measures of protein and provides measures of amino acid composition of samples but is far more expensive.
Protein Standard	Bovine serum albumin (BSA) vs immunoglobulin G (IgG) vs bovine gamma globulin (BGG)	Protein standards differ in amino acid content. Given that protein assays primarily react with only several amino acids, the choice of protein standard will affect the estimate of protein measured with the assay.	Most protein assays note conversion factors that can be used to convert protein measures estimated with one standard to an estimate based on another standard. Users could take the average of the estimate from BSA and IgG rather than choosing to present data based on one or the other standard.
Lipid Assay	Colorimetric	Some will only, or primarily, measure certain types of lipids (e.g. the sulfo-phospho-vanillin assay only detects unsaturated lipids).	First consider the size of the invertebrate. Colorimetric assays are the most practical solution for very small invertebrates (e.g., < 5 mg dry mass). Then consider what lipids you want to measure to address the goals of your study (e.g., a specific type or all lipids).
		May be better for life history studies in which users are interested in measuring specific types of lipids.
		May be used on any size of invertebrate, including individual collembolans or aphids.
	Gravimetric	Measures total lipid content, which can include triglycerides and phospholipids. This is a very easy assay, especially on larger invertebrates. This can be a better measure of nutrients available to consumers of an arthropod.
Carbohydrate Assay	Simple sugars	Not a common form of carbohydrate in insects, mainly found in sap or nectar feeding insects. Choice of standard (e.g., glucose vs. sucrose) may be important.	The user must consider the goals of the study, particularly the reason for measuring carbohydrates and which carbohydrates are most relevant to addressing the study question. The anthrone assay will detect simple sugars and will break down glycogen and trehalose, but other assays could be considered on a case-by-case basis for further applications.
	Glycogen and Trehalose	These are common forms in which carbohydrates are stored in insects.
Exoskeleton Determination		This assay measures the mass of exoskeleton present in an arthropod.	This may be useful to measure in studies of arthropod morphology or when measuring the quality of arthropods as food for predators since exoskeletal chitin is indigestible to most consumers and is equally unassimilated by predators with extra-oral digestion.

 

The aphid Metopolophium dirhodum (Walker, 1849; Hemiptera: Aphididae), house cricket Acheta domesticus (Linnaeus, 1758; Orthoptera: Gryllidae), German cockroach Blatella germanica Linnaeus, 1767 (Blattodea: Ectobiidae), mealworm larvae Tenebrio molitor Linnaeus, 1758 (Coleoptera: Tenebrionidae) and springtail Folsomia candida Willem, 1902 (Entomobryomorpha: Isotomidae) were used to test the protocol’s limits of detection, given their ease of cultivation and range of dry masses (in this study, mean ± SD, F. candida 1.14 ± 0.55 mg, M. dirhodum 3.10 ± 0.65 mg, A. domesticus 22.20 ± 5.83 mg, B. germanica 22.53 ± 4.96 mg, T. molitor 36.20 ± 22.30).

Samples were first weighed and lipids were extracted by soaking whole arthropods in 1 ml of 1:12 chloroform:methanol for 24 h (smaller specimens such as those <0.5 mg dry mass could be soaked in 0.5 ml for increased detectability, and larger specimens in larger volumes ~5x their body volume to ensure full submersion and to prevent saturation of the solvent). Half of the added volume of supernatant was then pipetted into a fresh tube for later lipid determination, the rest of the supernatant discarded, and any residue allowed to evaporate. This procedure for soaking arthropods was repeated for another 24 h, but discarding all supernatant, to ensure any residual lipids were removed from the sample prior to protein and carbohydrate extraction. The change in dry mass of a sample before and after soaking in the solvent can also be used as an estimate of the lipid content of samples where practicable (i.e. gravimetric assay).

Following the lipid assay, the soft tissue of samples was digested to facilitate quantification of protein and carbohydrates. This procedure only measures the macronutrient content of the soft tissue of arthropods and not any protein that may be bound in the chitinous matrix of the exoskeleton during sclerotization. Whole arthropods from 1 – 10 mg lean mass (i.e., mass after lipid extraction) were weighed, added to a microcentrifuge tube along with a stainless-steel bead (~3-7 mm diameter) and lysed at room temperature using a TissueLyser II (Qiagen, Hilden, Germany) for eight minutes at 30 Hz in two-minute increments. Larger samples were ground (e.g., bead beating or mortar and pestle) and an approximately 5 mg subsample was weighed into a clean tube. To each tube was added 1 ml of 0.1 M NaOH (or 0.5 ml for smaller specimens, e.g. <1 mg). Tubes were placed in a thermo-shaker at 80 °C and 250 RPM for 30 min, then removed and left at room temperature overnight (~16 h). Samples were centrifuged for 10 min at 13,000 RPM and 600 µl of supernatant pipetted into a separate tube for protein and carbohydrate determination. Supernatant was diluted prior to assaying such that the concentration of lean tissue (approximately 25–75 % protein for arthropods) was approximately 1 – 2 mg ml^-1 to allow protein values to fall within the range of the protein assay kit (most commercial protein assay kits can measure 0.025 – 2 mg ml^-1 protein). Dilution of supernatant or change in volume of NaOH used, along with the mass of sample used, must be accounted for in subsequent calculations of protein content.

Exoskeletal mass determination

The exoskeleton content of samples can also be measured, which may be of interest in morphological studies or those concerned with the nutritional quality of arthropods for consumers (Figure 2). A separate sample was used for this measurement in this study since lysis of tissues was carried out during macronutrient extraction to facilitate rapid dissolution of all soft tissues. Preliminary work suggests that exoskeleton measurements of lysed tissue result in lower values than measurements on intact arthropod bodies (Wilder unpublished). To maintain intact exoskeletons, the exoskeletal measurements instead included a second round of NaOH treatment and longer heated incubations. Exoskeletal measurement could theoretically be carried out on the same specimens used for macronutrient determination, but appropriate care must be taken to ensure that the soft tissue is appropriately dissolved; separate specimens should thus be used where possible. First, lipid should be completely extracted from the sample as described above. Then, the exoskeleton of the sample should be lightly cracked and 0.1 M NaOH (a volume approximately 5–10 times that of the sample) should be added to a vial with the sample. Samples should be heated for 2 h at 80 °C and then allowed to soak overnight after which the NaOH should be removed and discarded. Centrifugation may help move the exoskeleton to the bottom of the vial. An additional volume of NaOH is added to the tubes and allowed to soak for 24 h at room temperature, after which the NaOH can again be removed and discarded. Similar volumes of water should then be added to samples and removed twice to rinse any remaining NaOH from samples. Exoskeleton content is then the mass of sample remaining in the vial.

Macronutrient determination

Colorimetric assays were selected for the determination of macronutrients, given their ease-of-use and capacity for high-throughput assaying of samples in 96-well plates (Rodrı et al. 2008; Cheng et al. 2011). All absorbance measurements were obtained from a Tecan Infinity M200 Pro plate reader (Tecan Life Sciences, Männedorf, Switzerland) with Magellan v.7.1 software (Tecan 2011). For all assays, standard dilution series for calibration of absorbance readings consisted of 0-2 mg ml^-1 in nine increments (0, 0.025, 0.125, 0.25, 0.5, 0.75, 1, 1.5 and 2 mg ml^-1), with corn starch diluted in water, lard oil diluted in methanol and bovine serum albumin (BSA) diluted in water for carbohydrates, lipids and proteins, respectively. For each assay three repeats were taken from each sample and standard.

For determination of lipids, a sulfo-phospho-vanillin method adapted from Cheng et al. (2011) was used (Figure 3, Supporting Information 1). This method determines unsaturated lipid content; for total lipid content, gravimetric methods are the most appropriate option, but difficult for small invertebrates without specialised scales. Samples for lipid analysis comprised the initial supernatant taken after chloroform/methanol extraction.

 

Figure 3: Protocol for the determination of lipid content using the sulfo-phospho-vanillin method (Supporting Information 1). Figure created using Biorender.com.

Given the range of available protein assays, each with different benefits, the same samples from the five species analysed were put through two different protein-based colorimetric assays: bicinchoninic acid (BCA), and Lowry assays (Figure 4; Supporting Information 2). These assays followed the manufacturer protocols for BCA and Lowry assays. Samples for protein analysis comprised the supernatant taken after NaOH extraction.

 

Figure 4: Protocol for the determination of protein content using the BCA and Lowry methods (Supporting Information 2). Figure created using Biorender.com.

For carbohydrate determination, the anthrone method, originally proposed by Dreywood (1946), was adapted (Figure 5; Supporting Information 3). Samples for carbohydrate analysis comprised the final supernatant taken after NaOH extraction.

Figure 5: Protocol for the determination of carbohydrate content using the anthrone method (Supporting Information 3). Figure created using Biorender.com.

For each assay, the absorbance measurement of the blank standard was subtracted from all other absorbance measurements and a standard calibration curve prepared by plotting the blank-corrected measurement of the standards against their known concentrations. The regression equation of the standard curve was used to determine macronutrient concentration in each sample in mg ml^-1, which was then used to calculate the total macronutrient concentration in the sample based on the sample weight used for analysis and any dilution that was applied to the sample.

Limits of MEDI

Limit of blank (LoB) and limit of detection (LoD) describe the largest apparent concentration of analyte expected for blank samples and the lowest concentration likely to be detected and distinguished from a blank sample, respectively. The smallest detectable difference (SDD) is the smallest variance of measurement required to deem two measurements distinct. The LoB and LoD were determined as discussed by Armbruster and Pry (2008) from Clinical and Laboratory Standards Institute (2004), while the SDD as outlined by Kropmans et al. (1999) based on McNemar (1969). Calculations used the below equations where ‘B’, ‘SD’ and ‘SE’ denote concentration readings for 60 blank methanol samples taken from the same plate, standard deviation of those readings, and standard error of those readings, respectively.

LoB=mean_B + 1.645(SD_B)

LoD=LoB+1.645(SD_B)

SDD=1.96(2(SE))

 

Statistical analysis

All statistical analyses were performed using R version 3.5.3 (R Core Team 2020). To compare the macronutrient content of the species analysed, multivariate linear models (MLMs) were fitted using the ‘manylm’ function of the ‘mvabund’ package (Wang et al. 2012). Ternary plots were produced via ‘ggtern’ (Hamilton and Ferry 2018) and ‘ggplot2’ (Wickham 2016).

"ReadMe.txt" contains information regarding the other two files.

"MEDI Example Specimens.csv" contains data for each macronutrient (protein represented by the Lowry assay) and for the two protein assays trialled separately - Lowry and BCA. Mass columns denote the macronutrient mass detected, calculated per specimen. Percent mass indicates the percentage of total specimen body mass accounted for by macronutrient mass. Percent macros denotes the percentage of total macronutrient mass detected accounted for by each macronutrient.

"MEDI R script.R" contains the R scripts used to run the statisical analysis.