Dec 05, 2023

Public workspaceMetabarcoding based gut content analysis of arachnids

DOI
dx.doi.org/10.17504/protocols.io.5jyl8ppyrg2w/v1
  • 1Hebrew University of Jerusalem, Israel;
  • 2Newcastle University
Jordan P Cuff
Open access
DOI: dx.doi.org/10.17504/protocols.io.5jyl8ppyrg2w/v1
Protocol Citation: Yuval Zaltz, Evgenia Propistsova, Efrat Gavish-Regev, Jordan P Cuff 2023. Metabarcoding based gut content analysis of arachnids. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8ppyrg2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 24, 2023
Last Modified: December 05, 2023
Protocol Integer ID: 86904
Keywords: ecology, diet analysis, molecular dietary analysis, DNA metabarcoding, gut content analysis, spiders, scorpions, trophic interactions
Funders Acknowledgement:
Israel Science Foundation
Grant ID: 2656/20
Abstract
This protocol describes the workflow used for the gut content analysis via DNA metabarcoding of scorpions and spiders for some work conducted in the Hebrew University of Jerusalem, Israel. The protocol describes sample processing, DNA extraction, PCR amplification, gel electrophoresis, DNA normalisation, PCR-based library preparation, DNA purification and (briefly) sequencing and bioinformatics.
Image Attribution
Created with Biorender.
Materials
For field collection and initial storage:
  • Arachnid survey equipment (study specific)
  • Small collection tubes for storage of samples (e.g., 1.5 mL microcentrifuge tubes)
  • Sample labels (consider alcohol and freezer resistant labels, e.g., laser printed)
  • 100 % ethanol
  • Chemgene/diluted bleach for sterilisation of equipment

For DNA extraction:
  • Tissue homogeniser with sufficient number of 'heads' for running multiple samples in parallel
  • Qiagen DNeasy Blood and Tissue kit reagents and plasticware
  • 1.5 mL microcentrifuge tubes for final elution
For DNA amplification and subsequent steps:
  • Tagged PCR primers (with bridge primer sequence for Nextera library preparation by PCR if following that process)
  • 2X hot-start Taq polymerase mastermix (e.g., Qiagen Multiplex PCR kit or MyFi)
  • Molecular grade water
  • 96-well PCR plates
  • Mineral oil
  • 0.1X and 1X SPRI beads
  • Nextera Illumina adapter index primers (unless library prep to be completed by an external sequencing facility)
Buffers and reagents:
  • Sodium chloride
  • 1 M Tris-HCl
  • 0.5 M EDTA
  • Nuclease-free water
  • PEG
  • HCl
  • Tris
  • Guanidine HCl
  • 100 % ethanol
  • 1X TBE
  • Agarose
  • Gel stain (e.g., SYBRSafe)
  • DNA dye for gel electrophoresis
  • Gel ladder (50 bp - 500 bp)
Equipment:
  • -20 °C freezer
  • Tissue homogeniser or similar for homogenisation
  • Thermocycler
  • Magnetic stand (for plates and tubes)
  • Centrifuge
  • Microcentrifuge
  • Vortex
  • Pipettes (preferably including multichannel, ideally including 96-well)
  • Gel imaging dock
  • Gel tank and power pack
  • Illumina sequencer
Safety warnings
Attention
Check safety guidelines for individual reagents before commencing work. Some reagents will be toxic, corrosive or otherwise present health and safety risks. Appropriate personal protective equipment should be used at all times, not only for personal safety but also reduction of sample cross-contamination risk.
Ethics statement
The welfare of invertebrates, although poorly regulated, is of crucial importance when collecting and analysing them. Limiting suffering, but also taking only what is necessary is key. See the below citations for some considerations for this.

Crump A, Gibbons M, Barrett M, Birch J, Chittka L (2023) Is it time for insect researchers to consider their subjects’ welfare? PLoS Biol 21(6): e3002138. https://doi.org/10.1371/journal.pbio.3002138

Cuff, J. P., Barrett, M., Gray, H., Fox, C., Watt, A., & Aimé, E. (2023). The case for open research in entomology: reducing harm, refining reproducibility and advancing insect science. OSF Preprints https://doi.org/10.31219/osf.io/cufpq
Before start
Plan your experiment carefully. Ensure appropriate safety of all researchers throughout the work. Ensure welfare of the invertebrates used is appropriately considered. For the reduction of sample cross-contamination, consider using separate pre- and post-PCR work spaces and use bleach/Chemgene and/or UV decontamination of surfaces and equipment throughout.
Sample preparation
Sample preparation
2m
Collect arachnids from study sites, place them into Concentration100 % volume ethanol and store them at Temperature-20 °C until ready to proceed with DNA extraction.

Note
Consider the experimental design carefully to ensure that there is sufficient replication of the taxa sampled to answer the research questions. Consider the welfare and conservation of the invertebrate individuals and populations throughout this process too.

Depending on the arachnid, the optimal manner of extracting gut contents can differ. Here, we present two subtly different methods for scorpion and spider gut content analysis. These will be more broadly applicable to other arachnids (e.g., pseudoscorpions, mites, solifugids) but also many other invertebrates.
Step case

Isolation of gut contents from scorpions
68 steps

Using bleach-sterilised tools, cut the scorpion's abdomen open and scrape the innards of the abdomen into a 1.5 mL microcentrifuge tube.

Note
Given the size of scorpions used for this analysis, specific organs were not extracted, but see the citation below to guide selection of organs for optimal prey DNA prevalence.

CITATION
Simone, Y., Chaves, C., van der Meijden, A. & Egeter, B. (2022). Metabarcoding analysis of different portions of the digestive tract of scorpions (Scorpiones, Arachnida) following a controlled diet regime shows long prey DNA half-life. Environmental DNA.
LINK
https://doi.org/10.1002/edn3.311

2m
DNA extraction
DNA extraction
2h 15m 3s
DNA extraction follows the Qiagen DNeasy Blood & Tissue Kit protocol.

Add Amount180 µL Buffer ATL to the isolated tissue in the 1.5 mL microcentrifuge tubes.

Note
As per the Qiagen protocol, sub-sample tissue exceeding approximately Amount25 mg .


1m
Homogenise abdomens with a tissue homogeniser for approximately Duration00:00:03 or until fully homogenised.

Note
You can reuse the 'head' of the tissue homogeniser if appropriately sterilised between samples. For this, wash thoroughly in bleach, ensuring any remaining tissue is removed, then rinse in water and ethanol, and air-dry.

3s
Add Amount20 µL Proteinase K to tubes.

1m
Incubate on a thermoshaker at Shaker150 rpm, 56°C, 02:00:00 .

2h
Add Amount200 µL Buffer AL and vortex/shake to mix.

1m
Add Amount200 µL Concentration100 % (v/v) ethanol and vortex/shake to mix

1m
Pipette the mixture (~Amount600 µL ) into a spin column held within a 2 mL collection tube and centrifuge for Centrifigation6000 x g, 00:01:00 . Discard the flow-through and collection tube.

Note
Collection tubes can be cleaned and re-used if washed thoroughly with bleach. This reduces costs for additional extractions and reduces plastic waste.

2m
Place the spin column into a new collection tube, add Amount500 µL Buffer AW1 and centrifuge for Centrifigation6000 x g, 00:01:00 . Discard the flow-through and collection tube.

2m
Place the spin column into a new collection tube, add Amount500 µL Buffer AW2 and centrifuge for Centrifigation20000 x g, 00:03:00 . Discard the flow-through and collection tube.

4m
Place the spin column into a new 1.5 mL microcentrifuge tube, add Amount200 µL Buffer AE, incubate at TemperatureRoom temperature for Duration00:01:00 and centrifuge for Centrifigation6000 x g, 00:01:00 .


Note
You can repeat this step, eluting into the same tube, to increase mass yield, but the concentration of the DNA may be lower as a result.

3m
PCR amplification
PCR amplification
2h 47m
Decide how samples will be distributed across plates (but don't distribute DNA yet). Consider including a negative control in each row and column to detect any contaminants in each tagged forward and reverse primer. Among these wells, include any DNA extraction negative controls. Include positive controls (ideally mixed samples of species not found in the same study system), perhaps one adjacent to negative controls and the other adjacent only to samples (but both on separate rows and columns). Include blank controls (ideally wells into which no reagents or at least no primers are added), perhaps one adjacent to negative controls and the other adjacent only to samples (but both on separate rows and columns).

If using multiple PCR primer pairs, familiarise yourself with the annealing temperatures for each and prepare separate PCR plates for each. For optimal accuracy, consider running replicates of each reaction (e.g., triplicates).

Example layout of samples and controls in a 96-well plate.

Note
Consider your PCR primers carefully, ensuring your recovery of data is sufficient to answer your questions, particularly considering amplification of predator DNA (see the citation below).

CITATION
Cuff, J. P., Kitson, J. J. N., Hemprich-Bennett, D., Tercel, M. P. T., Browett, S. S., & Evans, D. M. (2023). The predator problem and PCR primers in molecular dietary analysis: Swamped or silenced; depth or breadth?. Molecular Ecology Resources.
LINK
https://doi.org/10.1111/1755-0998.13705

Distribute one drop of mineral oil into each well of the PCR plate (~Amount20 µL ).

Note
This can be achieved by taking a large volume of mineral oil into the pipette tip and then gently plunging so that a drop forms and falls from the tip into each well. Mineral oil improves sealing of reactions by preventing evaporation and condensation. This reduces evaporation and thus loss of product, but also reduces potential contamination.

2m
Prepare enough PCR mastermix for each sample.

For a full plate, these values should suffice (with some overage to account for pipetting error), but check your specific Taq polymerase mix for any differences:

AB
ReagentVolume (μL)
Molecular grade water (DNase free)1056
2X hot-start PCR mastermix1320

Note
These values are for Amount25 µL reaction volumes, which have been demonstrated to be effective for bulk samples. Consider running them in triplicate for more accurate results.


2m
For ease, if able to use a 96-well pipette, consider creating a "primer plate" containing both PCR primers for each well at Concentration2.5 micromolar (µM) concentration; this is especially effective when using multiple plates. Add Amount20 µL mastermix to each well of the PCR plate, followed by Amount2 µL of each primer mix from the primer plate to its corresponding well in the PCR plate. It is possible to use this strategy with a multi-channel or even single-channel pipette, but the alternative described immediately below may be easier.

If not using a 96-well pipette, consider making eight mastermixes each with sufficient reagents for 12 samples (Amount20 µL µL mastermix per well, so Amount264 µL µL for 12 with some overage for pipetting error), each containing a different forward primer (with Amount2 µL µL of Concentration2.5 micromolar (µM) forward primer per well, so Amount26.4 µL µL per mastermix for 12 with some overage for pipetting error). Distribute Amount22 µL µL of forward primer + mastermix to each well across the rows corresponding to each forward primer. Then distribute Amount2 µL of reverse primer to each well in the corresponding columns making sure to change pipette tip for each sample to avoid cross-contaminating the forward primers.

Distribution of tagged PCR primers across the 96-well plate

5m
Add Amount1 µL DNA to each corresponding sample and positive control well, and Amount1 µL molecular grade water to each negative control other than extraction negative controls (which should be added as samples).

5m
Briefly centrifuge the plate to ensure that the oil is above the PCR mix and everything is at the bottom of each well without air bubbles.
1m
Load the PCR plate into a thermocycler. Ensure that the temperature regime matches the enzyme used (including any heat activation for hot-start Taq) and that the annealing temperature matches the PCR primers used.

Note
Given differences between labs and sample material, and inaccuracies in temperature calibration, considering running a temperature gradient PCR with known samples to check optimal temperatures first if unsure.

2h 30m
The samples should now be checked for successful amplification, contamination in negatives and any secondary banding. Gel electrophoresis will achieve this, but digital systems like the Qiagen Qiaxcel will do this and facilitate equalisation by generating amplicon-specific DNA concentrations. Gel electrophoresis is nevertheless described below.
Gel electrophoresis
Gel electrophoresis
1h 15m
Prepare a gel tank containing sufficient 1X TBE buffer to submerge the gel.

Note
TBE buffer can be purchased pre-made or made from its constituent reagents. Alternative buffers like borax can, however, be cheaper, easier and quicker to use.

1m
Prepare a Concentration2 Mass / % volume agarose gel for the samples to go into (detailed in the below substeps).

Prepare a gel mould with sufficient comb tips for the number of samples to be run. Ensure these are spaced out enough to allow the gel to run and separate without running into another well.
1m
Determine a suitable size (and therefore volume) of gel based on the size of the tank available. For a Amount150 mL gel, weigh Amount3 g of agarose and add it to Amount150 mL of 1X TBE buffer.

2m
Heat this mixture in a microwave in a conical flask until it begins bubbling.
2m
Mix the solution by gently swirling it and heat again until bubbling. Repeat this as necessary until there are no large lumps of agarose remaining.

Note
It can be beneficial to let the mixture cool for Duration00:00:30 before proceeding.


1m
Add Amount3 µL of gel dye (e.g., SYBRSafe) and mix by swirling.

1m
Pour the mixture into the gel mould and allow to set for Duration00:25:00 .

25m
Add Amount2 µL of each sample to a new plate and add Amount2 µL of 1X DNA dye for visualisation of the products in the gel.

10m
Once set, place the gel into the tank and add Amount4 µL of DNA gel ladder to each side of the gel in each row, and then Amount4 µL of dyed PCR product to each other well.

Note
Ensure that the DNA will run in the correct direction when placing the gel in the tank. TIP: it will 'run to the red'.


5m
Once filled, run the gel power pack at 120 V and 120 A for Duration00:25:00 .

25m
Visualise the gel under a UV or blue light in a gel dock to confirm amplification and a lack of contamination in negative controls.

Note
Ensure you are not exposed to UV by only using the UV light in a contained dock and/or with appropriate protective equipment.

2m
PCR product normalisation
PCR product normalisation
1h 21m
If the PCRs were replicated (i.e., each sample run multiple times for each PCR primer pair used), these can be merged together into one plate per PCR primer pair at this point, or carried forward separately. Keeping the replicates separate increases the number of libraries to prepare and sequence later, but better facilitates identification of inconsistencies between samples. To merge triplicates, assuming use of Amount25 µL reaction volumes, pipette Amount20 µL from each well of two of the three plates into the corresponding well of the third. Briefly centrifuge the merged plate to move the oil to the top of the product again.

Note
To avoid pipetting oil from the oil-sealed PCR products, plunge the pipette to the first stop and fully insert the pipette tip into the bottom of the well, then release sharply. The PCR product will be taken up quickly, whereas the relatively viscous oil will be taken up slowly, thus being outcompeted by the PCR product and mostly left behind.

5m
Prepare 0.1X SPRI bead solution and bring to room temperature. The below steps detail how to make this solution, but it is also commercially available.

If using beads such as Sera-Mag Magnetic SpeedBeads (carboxylated, Thikness1 µm , 3 EDAC/PA5), take Amount1 mL of well-mixed bead solution and wash the beads twice with TE+Tween buffer (Concentration10 millimolar (mM) Tris base, Concentration1 millimolar (mM) EDTA, Concentration0.05 % volume Tween 20, Ph8.0 ) by magnetising the beads, removing the supernatant, adding the TE+Tween, remagnetising the beads and removing the supernatant, and repeating the addition and removal of TE+Tween once more.

5m
To the beads, add the following mix:

AB
ReagentVolume
5 M NaCl25 mL
Molecular grade water3.582 mL
1 N HCl0.168 mL
1 M Tris base0.5 mL
0.1 M disodium EDTA0.5 mL

5m
Add Amount20 mL of Concentration50 % volume PEG to the tube to reach a 1X bead solution (alongside making the 0.1X solution, this will be useful later).

1m
Add 5 mL of 1X bead solution to Amount45 mL of the following mix to make a 0.1X solution:

AB
ReagentVolume
5 M NaCl25 mL
Molecular grade water3.582 mL
1 N HCl0.168 mL
1 M Tris base0.5 mL
0.1 M disodium EDTA0.5 mL
50 % PEG20 mL

1m
If ≥Amount20 µL of PCR product is available in each well, pipette Amount20 µL of 0.1X SPRI bead solution into each well of a new 96-well plate. If less PCR product is available, add a volume equivalent to the available PCR product volume of 0.1X SPRI beads to a new 96-well plate.

Note
When working with magnetic beads, ensure they are fully mixed with no residue at the bottom of the container.

2m
Add Amount20 µL of PCR product (or whatever volume of beads was used in the last step) to each corresponding well of 0.1X beads, avoiding oil, and mix by vortexing (Shaker1500 rpm, 00:01:00 ).

Note
To avoid pipetting oil from the oil-sealed PCR products, plunge the pipette to the first stop and fully insert the pipette tip into the bottom of the well, then release sharply. The PCR product will be taken up quickly, whereas the relatively viscous oil will be taken up slowly, thus being outcompeted by the PCR product.

3m
Incubate at TemperatureRoom temperature for Duration00:05:00 .

5m
Place on a magnetic stand for Duration00:05:00 .

5m
Remove all but Amount5 µL of the mixture from each well via pipette without disturbing the beads, which should be settled on the magnet (although can be hard to see with 0.1X bead solutions).

2m
Add Amount200 µL Concentration80 % volume ethanol to each well.

1m
Remove the ethanol and add a further Amount200 µL Concentration80 % volume ethanol to each well.

2m
Remove the ethanol as completely as possible by pipetting.

Note
A small amount of remaining ethanol isn't debilitating in this case, unlike usual magnetic bead cleaning protocols, because we will complete a subsequent purification immediately after.

1m
Add Amount10 µL molecular grade water to each well, shake at Shaker1500 rpm, 00:01:00 and incubate at TemperatureRoom temperature for Duration00:05:00 .

7m
Place on a magnetic stand for Duration00:05:00 .

5m
Pool Amount8 µL from each well into a single tube, leaving the magnetic beads behind in the 96-well plate.

Note
To pool samples using a multi-channel pipette, pipette each column of samples into a new strip of Amount200 µL tubes, then merge all of these tubes into a single 1.5 mL tube at the end.


2m
Mix the pooled samples by flicking and inverting, and transfer Amount700 µL into a new 1.5 mL microcentrifuge tube.

1m
Add Amount700 µL 1X magnetic beads for DNA purification (see note below for size selection) and mix by vortexing. Incubate at TemperatureRoom temperature for Duration00:05:00 .

Note
This is a good opportunity for size selection as well, especially if the PCR product contains any secondary bands. If using amplicons around 350 bp, consider a 0.8X bead ratio (i.e., add Amount560 µL beads instead).


6m
Place on a magnetic stand for Duration00:05:00 .

5m
Add Amount400 µL Concentration80 % volume ethanol.

1m
Remove the ethanol and add a further Amount400 µL Concentration80 % volume ethanol.

1m
Remove the ethanol completely. Briefly centrifuge the sample to collect and remove any residual ethanol. Allow to air-dry until the aggregation of magnetic beads transitions from 'glossy' (shiny reflection of light) to 'matte' (dull dark brown mass), but not so long that it dries completely (i.e., begins to turn a rusty red and shows cracks).
3m
Add Amount100 µL molecular grade water and mix by vortexing. Incubate at TemperatureRoom temperature for Duration00:05:00 .

6m
Place the sample on a magnetic stand for Duration00:05:00 .

5m
Remove Amount95 µL of the water and place it in a new tube. This will be the library used in the subsequent section.

1m
PCR2 and library preparation
PCR2 and library preparation
2h 21m
Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).

Note
At this point, many sequencing facilities will accept the samples and can carry out the subsequent steps themselves. This may be preferable if the appropriate library preparation/indexing primers are not available. Below, the process for PCR-based library preparation will be detailed as an example.

5m
Dilute library to ≤Concentration5000 µg/µL (≤5 ng/µL) in molecular grade water.

Note
Without diluting the library, there is a risk that the adapters will not be added to all DNA (i.e., there is too much DNA) which will compromise the sequencing yield.

1m
Add a drop of mineral oil to one PCR tube for each library, alongside an additional tube for a negative control.
1m
Per library, assemble the following reaction:

AB
ReagentVolume
2X hot-start PCR mastermix7.5
Molecular grade water1
Nextera sequencing adapter primer mix1.5
DNA library5

5m
Briefly centrifuge the tubes to ensure that the oil is above the PCR mix.
1m
Load the PCR reactions into a thermocycler. Ensure that the temperature regime matches the enzyme used (including any heat activation for hot-start Taq) and that the annealing temperature matches the PCR primers used. Run for 12 cycles.
1h 30m
Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
5m
Check that the adapters have been added to the DNA by determining and comparing the amplicon sizes for libraries before and after this second PCR. Amplicons should be longer after the second PCR. This is ideally assessed using a digital system like TapeStation, but even gel electrophoresis will work.
30m
Once successful adapter addition is confirmed, libraries can be pooled so that each is equimolar in the final mixture based on the concentrations determined above (e.g., by Qubit).

Note
This can be achieved by dividing the maximum concentration across the libraries by each library concentration and pooling that many µL from each. These values may need to be multiplied up to ensure adequate yield for sequencing.
Be careful if using multiple PCR primer pairs! Different amplicons have different molecular weights, so pool based on fmol rather than ng/µL.

3m
Sequencing
Sequencing
Check with your sequencing provider how many fmol they will need in how many µL. This will be based on the sequencer, sequencing cartridge and any QC processes they follow. The libraries should be ready for sequencing. Sequencing times will depend on the machines, protocols and any queues involved at the sequencing facility.
Bioinformatics
Bioinformatics
Optimal bioinformatics workflows depend on a variety of factors including target taxa, study system, the genetic marker used, computational resources available and various other factors. Other resources with code integration will be much better suited to detailing the steps involved. The process will likely follow a similar progression of steps though:

  1. Read processing: demultiplex, trim primers, merge pairs.
  2. Filtering and error correction: quality filtering, dereplication, denoising, chimera filtering.
  3. Taxonomic unit aggregation: OTU/ASV delimitation.
  4. Taxon assignment: reference database curation, taxon assignment.
Protocol references
Simone, Y., Chaves, C., van der Meijden, A. & Egeter, B. (2022). Metabarcoding analysis of different portions of the digestive tract of scorpions (Scorpiones, Arachnida) following a controlled diet regime shows long prey DNA half-life. Environmental DNA, 4, 1176–1186. https://doi.org/10.1002/edn3.311

Macías-Hernández, N., Athey, K., Tonzo, V., Wangensteen, O.S., Arnedo, M., Harwood, J.D. (2018). Molecular gut content analysis of different spider body parts. PLoS ONE 13(5): e0196589.
https://doi.org/10.1371/journal.pone.0196589

Cuff, J. P., Kitson, J. J. N., Hemprich-Bennett, D., Tercel, M. P. T., Browett, S. S., & Evans, D. M. (2023). The predator problem and PCR primers in molecular dietary analysis: Swamped or silenced; depth or breadth? Molecular Ecology Resources, 23, 41–51. https://doi.org/10.1111/1755-0998.13705
Citations
Step 14
Cuff, J. P., Kitson, J. J. N., Hemprich-Bennett, D., Tercel, M. P. T., Browett, S. S., & Evans, D. M.. The predator problem and PCR primers in molecular dietary analysis: Swamped or silenced; depth or breadth?
https://doi.org/10.1111/1755-0998.13705
Step 14
Cuff, J. P., Kitson, J. J. N., Hemprich-Bennett, D., Tercel, M. P. T., Browett, S. S., & Evans, D. M.. The predator problem and PCR primers in molecular dietary analysis: Swamped or silenced; depth or breadth?
https://doi.org/10.1111/1755-0998.13705
Step 3
Macías-Hernández, N., Athey, K., Tonzo, V., Wangensteen, O.S., Arnedo, M., Harwood, J.D.. Molecular gut content analysis of different spider body parts
https://doi.org/10.1371/journal.pone.0196589
Step 3
Simone, Y., Chaves, C., van der Meijden, A. & Egeter, B.. Metabarcoding analysis of different portions of the digestive tract of scorpions (Scorpiones, Arachnida) following a controlled diet regime shows long prey DNA half-life
https://doi.org/10.1002/edn3.311