TITLE: MEDI: Macronutrient Extraction and Determination from Invertebrates
AUTHORS: Jordan Cuff, Shawn M. Wilder

[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 ~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.</div></div>

[STEPS]
?. [Collection and preparation of materials]
Collect invertebrates and kill them, ideally by freezing.
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) to ensure all solubilised material is accessible for downstream analysis. This can be achieved during the drying described in step 2.
?. [Determination of lipid content]
Prepare a stock standard dilution series using a suitable analogue (e.g. analytical lard oil) of known concentration diluted with 1:12 chloroform:methanol. A dilution series of 0-2 mg/ml in nine increments (0, 25, 125, 250, 500, 750, 1000, 1500 and 2000 μg/ml) should suitably cover a good range of concentrations, but this can be adjusted accordingly.
?. [Determination of protein and carbohydrate content]
From the original tube containing the remaining chloroform:methanol solution and the invertebrate tissue, discard the remaining supernatant (having already removed 500 μl for lipid analysis) avoiding the removal of any tissue. Add another 1 ml 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.
?. [Collection and preparation of materials]
Weigh the dried invertebrate using appropriately sensitive scales, recording the mass.
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, particularly if using gravimetric estimation of lipid content.
If the invertebrate mass is greater than 10 mg, a gravimetric measurement of lipid (detailed below) will likely be more accurate, and following lipid analysis it will be necessary to sub-sample the remaining tissue. If the invertebrate is less than 10 mg, proceed with colorimetric assays without sub-sampling.
?. [Collection and preparation of materials]
Desiccate the invertebrate specimens via freeze-drying 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.
?. [Determination of exoskeletal mass]
For the greatest accuracy, exoskeletal mass determination should be carried out in parallel to the macronutrient analyses with different specimens, but it is possible to  streamline these together if necessary. If you do wish to streamline them, the protocol for exoskeletal mass determination largely follows the steps for the macronutrient determination, so attention will be drawn to the overlapping aspects. In order to streamline these processes, exoskeletal determination should take place after extraction of samples for lipid, and carbohydrate and protein determination. When lysing the tissue for protein and carbohydrate determination, the exoskeleton should instead simply be cracked to facilitate penetration of the tissue by the NaOH without breaking apart the exoskeleton itself.
?. [Determination of exoskeletal mass]
Add 1 ml 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 can be designed to overlap with STEP 17 of "Determination of lipid content".
If working with very large samples (e.g. >50 mg), it is recommended to add ~5X the body volume of the invertebrate.
Different ratios of chloroform and methanol can be used (e.g. 2:1, 1:9), including pure chloroform. It is advised to consistently use the same ratio.
?. [Welcome to MEDI!]
Welcome to Macronutrient Extraction and Determination from Invertebrates! Here's an overview of the extraction protocol:There are several overnight incubation steps, so don't be alarmed by some of the long procedure times!
?. [Determination of lipid content]
Add 1 ml 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.
If working with very small samples (e.g. If working with very large samples (e.g. >50 mg), it is recommended to add ~5X the body volume of the invertebrate, the volume of which must be recorded and factored into the subsequent calculations of lipid content.
?. [Determination of exoskeletal mass]
Discard the supernatant, avoiding the removal of any tissue, add another 1 ml 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 can be designed to overlap with STEP 28 of "Determination of protein and carbohydrate content". The removal of residual lipids is necessary for the accurate analysis of protein content given the inhibition of protein assays by lipids.
?. [Determination of exoskeletal mass]
Discard the supernatant, avoiding the removal of any tissue, and evaporate any remaining residual solvent.
This can be designed to overlap with STEP 29 of "Determination of protein and carbohydrate content".
?. [Determination of exoskeletal mass]
Add 0.1 M NaOH to the sample tube, aiming for 5-10X the body volume of the invertebrate. Lightly crack the exoskeleton to expose the inner tissues, and incubate at 80°C for 2 h.
This can be designed to overlap with STEP 31 of "Determination of protein and carbohydrate content", but with an undefined volume of solvent (rather than 1 ml), cracking of the exoskeleton rather than lysis, and increased incubation time.
?. [Determination of exoskeletal mass]
Allow the sample to soak overnight at room temperature.
This can be designed to overlap with STEP 32 of "Determination of protein and carbohydrate content".
?. [Determination of exoskeletal mass]
Discard the supernatant, avoiding the removal of any tissue. Add a further round of 0.1 M NaOH (the same volume) to the sample tube, and incubate at 80°C for 2 h.
Lightly centrifuge to move the exoskeletal fragments to the bottom of the tube if necessary.
?. [Determination of exoskeletal mass]
Allow the sample to soak overnight at room temperature.
?. [Determination of exoskeletal mass]
Discard the supernatant, avoiding the removal of any tissue. Add water to the tube (the same volume as with NaOH) and remove (without removing any exoskeletal mass) to wash away any remaining NaOH.
?. [Determination of exoskeletal mass]
Repeat the water wash in the previous step so that it has been carried out twice in total.
?. [Determination of exoskeletal mass]
Remove the water (without removing any exoskeletal mass) and evaporate any remaining water.
?. [Determination of exoskeletal mass]
Weigh the remaining exoskeleton in the tube and subtract the mass of the tube to calculate the exoskeletal mass.
?. [Determination of lipid content]
The lipid content of the 500 μl of chloroform:methanol solution removed during STEP 18 can be determined using the sulfo-phospho-vanillin method.
?. [Determination of lipid content]
Remove 500 μl of chloroform:methanol solution and keep it in a separate tube for lipid analysis. Unless the invertebrate dry body mass is >10 mg, the remaining solution in the original tube is taken forward for determination of protein and carbohydrate content (STEP 31), ideally with immediacy given additional overnight steps. If the body mass is >10 mg, the sub-steps below detail gravimetric lipid determination.
Ensure the tube containing the lipids is air tight to avoid evaporation, which will affect downstream concentration values. 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.
?. [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]
Add 1 ml 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.
If working with very small samples (e.g.
?. [Determination of lipid content]
From the original tube containing the remaining chloroform:methanol solution and the invertebrate tissue, discard the remaining supernatant (having already removed 500 μl for lipid analysis) avoiding the removal of any tissue. Add another of the same volume of 1:12 chloroform:methanol as in STEP 17 to each sample tube and leave the tube containing the specimen and chloroform:methanol at room temperature for 24 hours.
?. [Determination of lipid content]
Discard the supernatant, avoiding the removal of any tissue, and evaporate any remaining residual solvent.
?. [Determination of lipid content]
Weigh the remaining body mass and subtract it from the original dry mass to determine total lipid mass gravimetrically.
?. [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 (unless the dry body mass exceeds 10 mg, in which case follow the below sub-step).
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]
If working with invertebrates with a dry body mass >10 mg, lyse/homogenise the body and take a 3-7 mg subsample forward for the subsequent steps.
Take note of the mass of tissue taken forward. The subsequent absorbance readings will only represent this mass of tissue and must be multiplied to calculate the total protein/carbohydrate content of the whole specimen.
With 3-7 mg of dry tissue, dilutions will still likely be necessary downstream (detailed in STEP 35). This must also be accounted for in any calculations of total protein/carbohydrate content (i.e. the values will be for 3-7 mg of the specimen rather than its full mass).
?. [Determination of protein and carbohydrate content]
Allow the sample to soak overnight at room temperature.
This overnight step can be left out if ensuring the tissue is appropriately lysed, for example by including sonication in STEP 31 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]
Take 600 µl of the supernatant into a separate tube for taking forward for protein and carbohydrate determination.
?. [Determination of protein and carbohydrate content]
If possible, estimate the protein and carbohydrate content of the invertebrates and dilute accordingly for each assay to take forward 0.5-1.75 mg/ml.
If using 3-7 mg of dry tissue, as per STEP 30.1, a 1 in 5 dilution will likely be appropriate (i.e. 100 μl of sample with 400 μl NaOH). Remember to factor any dilutions into the final calculation of protein.
?. [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]
Measure absorbance at 490 nm using a spectrophotometer.
?. [Determination of protein and carbohydrate content]
Cool the plate to room temperature and measure absorbance at 620 nm using a spectrophotometer.
?. [Determination of lipid content]
Add 10 µl concentrated sulfuric acid to the lipid residues and vortex/mix before incubating again at 100 °C for 10 min.
?. [Determination of lipid content]
After 5 min, transfer 200 µl of each well into a new plate.
This is mostly in case the plate has warped at all during the heating stage, but also to remove any potential for particulate matter that may have precipitated during the initial evaporation.
?. [Determination of lipid content]
Allow the samples to cool to room temperature and add 240 µl vanillin reagent to each well, vortexing/mixing for homogenous colouration.
?. [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):ReagentAmountVanillin330 mgHot water220 μl85% phosphoric acid55 μl
ReagentAmountVanillin330 mgHot water220 μl85% phosphoric acid55 μl
?. [Determination of lipid content]
If possible, estimate the lipid content of the invertebrates and dilute accordingly to take forward a sub-sample of 0.5-1.75 mg/ml.
?. [Determination of protein and carbohydrate content]
The carbohydrate content of the 600 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 0-2 mg/ml in nine increments (0, 25, 125, 250, 500, 750, 1000, 1500 and 2000 μg/ml) should suitably cover a good range of concentrations, but this can be adjusted accordingly.
?. [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):ReagentAmountAnthrone185 mgConcentrated sulfuric acid185 μl
ReagentAmountAnthrone185 mgConcentrated sulfuric acid185 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 160 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 10 min.
?. [Determination of protein and carbohydrate content]
The protein content of the 600 μl of NaOH solution removed during STEP 34 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 summarised in the image below. This protocol will describe the protocol for the Lowry assay. The reagents involved in each of these assays are widely commercially available.
?. [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 0-2 mg/ml in nine increments (0, 25, 125, 250, 500, 750, 1000, 1500 and 2000 μg/ml) should suitably cover a good 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 different standards 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.