TITLE: Further Micro-scaled MEDI (Macronutrient Extraction and Determination from Invertebrates)
AUTHORS: Jordan Cuff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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 is a protocol for the direct, rapid and relatively low‐cost determination of macronutrient content from single small macroinvertebrates.</div><div class = "text-block">Macronutrients are 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.</div><div class = "text-block">Macronutrient Extraction and Determination from Invertebrates (MEDI) can directly and rapidly determine macronutrient content in tiny (dry mass <1 mg). 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.</div></div>

[STEPS]
?. [Welcome to MEDI!]
Welcome to Macronutrient Extraction and Determination from Invertebrates (further micro-scaled edition)! The following presents a micro-scaled version of the original MEDI protocol. The further micro-scaled protocol is intended particularly for invertebrates of dry volumes of 1 mg or less which cannot be pooled together given their scarcity or the focus of the study precluding pooling (e.g., investigation of individual differences). The micro-scaling is achieved by increasing analyte concentration at both the extraction and analysis stages, therefore facilitating more sensitive detection of macronutrients. Analytical standards must also be adjusted accordingly (detailed within).Here's an overview of the further micro-scaled extraction protocol:This is based on the protocol originally published in Methods in Ecology and Evolution:Unlike the original MEDI protocol, this protocol does not include the determination of exoskeletal mass which may be an important factor in many studies. This could theoretically be incorporated into the protocol according to the instructions in the original protocol, but the miniscule masses that this further micro-scaled protocol is designed for would preclude the mass-based measurements except where access to highly specialised scales is possible.There are several overnight incubation steps, so don't be alarmed by some of the long procedure times!
?. [Collection and preparation of materials]
Collect invertebrates and kill them, ideally by freezing or another optimally humane method.
If working with fluid-preserved specimens (e.g. alcohol-fixed), transfer all material including preserving agent into a suitable tube (e.g. 1.5 ml microcentrifuge tube or 2 ml screw-top collection tube) and evaporate the preservative (if possible/appropriate/safe) to ensure all solubilised material is accessible for downstream analysis. This can be achieved during the drying described in step 3.
?. [Collection and preparation of materials]
Desiccate the invertebrate specimens via freeze-drying (ideal) or heat-drying until completely dry.
Heat-drying can be carried out in a 60 °C oven for 24-48 hours, or a shorter time period if using a heated centrifuge with a vacuum pump. Be wary of burning or otherwise damaging the bodies.
?. [Collection and preparation of materials]
If possible, weigh the dried invertebrate using appropriately sensitive scales, recording the mass.
This weighing is useful for representation of macronutrient contents as a proportion of total body mass, but the values can alternatively be presented as proportions in the absence of body mass (although this is sub-optimal).
If the invertebrate was fluid-preserved and the preserving agent has been evaporated into the sample tube, ensure you measure the mass of the tube and any precipitated matter it contains as this will be important later.
?. [Determination of lipid content]
Add 500 μl of 1:12 chloroform:methanol to each sample tube and leave the tube containing the specimen and chloroform:methanol at room temperature for 24 hours.
?. [Determination of lipid content]
Remove 400 μl of chloroform:methanol solution and keep it in a separate tube for lipid analysis. The remaining solution in the original tube is taken forward to the next section (determination of protein and carbohydrate content; STEP 15), ideally with immediacy given that the next step is another overnight soak in chloroform:methanol.
Ensure the tube containing the lipids is air tight to avoid evaporation, which will affect downstream concentration values. Consider placing the tubes in a freezer to further reduce evaporation. Importantly, the tube must also be suitable for the storage of chloroform (i.e. won't dissolve) for which glass is ideal. To avoid potential removal of tube labels written in pen, paper with pencil writing on it can be left in the solution within the tube so long as risk of cross-contamination with lipids is mitigated (i.e., the paper is free of even residual lipids).
?. [Determination of lipid content]
The lipid content of the 400 μl of chloroform:methanol solution removed during STEP 6 can be determined using the sulfo-phospho-vanillin method.
?. [Determination of lipid content]
Prepare a stock standard dilution series using a suitable analogue to the lipids present in your tissues (e.g. analytical lard oil for animals) of known concentration diluted with 1:12 chloroform:methanol. A dilution series of 0-1 mg/ml in nine increments (0, 12.5, 62.5, 125, 250, 375, 500, 750 and 1000 μg/ml) should suitably cover a broad range of concentrations, and can be adjusted further according to expected concentrations.
?. [Determination of lipid content]
Make up the vanillin reagent using the following amounts (which should account for reagent overage) per sample/standard repeat (so thrice per sample/standard if running triplicates, as advised):ReagentAmountVanillin275 mgHot water183 μl85% phosphoric acid46 μl
ReagentAmountVanillin275 mgHot water183 μl85% phosphoric acid46 μl
?. [Determination of lipid content]
From each standard and sample, put three repeats of 50 µl into a flat-bottomed 96-well plate and heat at 100 °C in a ventilated hood until all solvent has evaporated (leaving just the lipid residue; ~10 min).
?. [Determination of lipid content]
Add 15 µl concentrated sulfuric acid to the lipid residues and vortex/mix before incubating again at 100 °C for 15 min.
This is one and a half times longer than in the original protocol. Leaving concentrated acids at high  temperatures presents a number of possible hazards. Remain vigilant and keep close watch of the plate.
?. [Determination of lipid content]
Allow the samples to cool to room temperature and add 200 µl vanillin reagent to each well, vortexing/mixing for homogenous colouration.
?. [Determination of lipid content]
After 5 min, transfer 200 µl from each well into a corresponding well in a new plate.
This is mostly in case the plate has warped at all during the heating stage, but also to remove any potential particulate matter that may have precipitated during the initial evaporation.
?. [Determination of lipid content]
Measure absorbance at 490 nm using a spectrophotometer.
?. [Determination of protein and carbohydrate content]
This continues with material generated in STEP 6.From the original tube containing the remaining chloroform:methanol solution and the invertebrate tissue, discard the remaining ~100 μl supernatant (having already removed 400 μl for lipid analysis) avoiding the removal of any tissue. Add another 500 μl of 1:12 chloroform:methanol to each sample tube and leave the tube containing the specimen and chloroform:methanol at room temperature for 24 hours.
This additional chloroform treatment is purposed to remove any residual lipids which can interfere with the downstream analyses.
?. [Determination of protein and carbohydrate content]
Discard the supernatant, avoiding the removal of any tissue, and evaporate any remaining residual solvent.
?. [Determination of protein and carbohydrate content]
Lyse the tissue and ensure it is mixed/homogenous. Lysis can be carried out using a pestle and mortar, or bead-beating method, ensuring all available tissue is taken forward and any beads added are removed.
If using a bead-beating method (e.g. Qiagen Tissuelyser II at 30 Hz for 8 min), it may be easier to do so in a liquid matrix, so you can do this after the initial addition of NaOH in the next step. Care must be taken to avoid contact with NaOH when doing so.
?. [Determination of protein and carbohydrate content]
Add 500 μl 0.1 M NaOH to the sample tube. Incubate at 80°C for 30 min in a themo-shaker to ensure mixture of the lysed material with the NaOH.
?. [Determination of protein and carbohydrate content]
Allow the sample to soak overnight at room temperature.
This overnight step can be left out if sure that the tissue is appropriately lysed, for example by including sonication and mixing halfway through.
?. [Determination of protein and carbohydrate content]
Centrifuge the sample for 10 min at 13,000 rpm.
?. [Determination of protein and carbohydrate content]
Transfer 400 µl of the supernatant into a separate tube for taking forward to protein and carbohydrate determination.
?. [Determination of protein and carbohydrate content]
The carbohydrate content of the 400 μl of NaOH solution removed during STEP 34 can be determined using the anthrone method.
?. [Determination of protein and carbohydrate content]
Prepare a stock standard dilution series using a suitable analogue (e.g. analytical corn starch) of known concentration diluted with polished water. A dilution series of 0-0.02 mg/ml in nine increments (0, 0.25, 1.25, 2.5, 5, 7.5, 10, 15 and 20 μg/ml) should suitably cover a range of minute concentrations, but this can be adjusted accordingly. These concentrations were used for analyses of relatively carbohydrate-poor taxa like parasitoid wasps, thus more concentrated standards, e.g., a factor of ten more, may be better for other studies. Carbohydrate is typically much less concentrated in many invertebrate tissues compared to protein and lipid.
?. [Determination of protein and carbohydrate content]
Make up the anthrone reagent using the following amounts (which should account for reagent overage) per sample/standard repeat (so thrice per sample/standard if running triplicates, as advised):ReagentAmountAnthrone165 mgConcentrated sulfuric acid165 μl
ReagentAmountAnthrone165 mgConcentrated sulfuric acid165 μl
?. [Determination of protein and carbohydrate content]
From each standard and sample, put three repeats of 40 µl into a flat-bottomed 96-well plate, add 140 µl anthrone reagent to each and vortex/mix.
?. [Determination of protein and carbohydrate content]
Incubate the plate at 92 °C in a ventilated hood for 30 min.
This is three times longer than in the original protocol. Leaving concentrated acids at high temperatures presents a number of possible hazards. Remain vigilant and keep close watch of the plate.
?. [Determination of protein and carbohydrate content]
Cool the plate to room temperature and incubate for a further 30 min to facilitate further development of the assay.
?. [Determination of protein and carbohydrate content]
The protein content of the 400 μl of NaOH solution removed earlier can be determined using one of several colorimetric protein methods. The three highlighted in the MEDI manuscript (the benefits and drawbacks also discussed therein) are all viable, as are several other alternatives. This protocol will describe the protocol for the Lowry assay.
?. [Determination of protein and carbohydrate content]
Prepare a stock standard dilution series using a suitable analogue (e.g. bovine serum albumin) of known concentration diluted with polished water. A dilution series of 0-1 mg/ml in nine increments (0, 12.5, 62.5, 125, 250, 375, 500, 750 and 1000 μg/ml) should suitably cover a broad range of concentrations, but this can be adjusted accordingly.
Most protein assay kits will allow values to be converted for different standards using standard conversion factors since variation in the standards used will differentially affect the results of each assay.
?. [Determination of protein and carbohydrate content]
From each standard and sample, put three repeats of 40 µl into a flat-bottomed 96-well plate, add 200 µl of Modified Lowry Reagent to each and vortex/mix.
?. [Determination of protein and carbohydrate content]
Incubate at room temperature (~20-25 °C) for 10 min.
?. [Determination of protein and carbohydrate content]
Add 20 µl of 1X (1N) Folin-Ciocalteu reagent to each well and vortex/mix.
?. [Determination of protein and carbohydrate content]
Incubate at room temperature (~20-25 °C) for 30 min.
?. [Determination of protein and carbohydrate content]
Measure absorbance at 750 nm using a spectrophotometer.
?. [Determination of protein and carbohydrate content]
Measure absorbance at 620 nm using a spectrophotometer.
?. [Analysis of absorbance readings]
You can now calculate the concentration of analyte based on the absorbance measurements. Keep in mind that the use of only 500 µl in each of the extraction phases means that mg/ml data will need dividing by two in order to calculate whole body macronutrient content. Any dilutions or other alterations to assay input concentrations/volumes must also be considered here.