Sample homogenization and DNA extraction for bulk insect catches

Start by creating individual working stations for each insect bottle that you will process by dividing the lab bench into 30 cm wide working stations. Number each station with a running number from 1 to n+1 (n is the total number of samples you will process) to account for one station to process your blank sample.

Use a squirt bottle with bleach 10% solution and a paper towel to wipe all lab benches.

At each working station place the following:

• 1 tray
• 1 bottle for temporary storage of homogenate (samples with ≥10 gr of insect biomass only)
• 500 mL Nalgene HDPE bottle (for the blank sample)
• small tweezers
• large tweezers
• large petri dish
• microscope
• silicon spatula (200 mm long - Fig. 1)
• silicon corner spatula (200 mm long - Fig. 1)
• 1 large glass petri dish
• IKA DT-50 rotor stator tube (50 mL)
• 1 screw cap microtube (2.0 mL)

Place one drained insect bottle in each working station and one empty bottle (Nalgene, 500 ml HDPE wide mouth) in the last working station ( n+1) working station (blank sample).

Using a marker pen, label each bulk insect bottle and rotor stator tube with the respective working station number.

If you have a nylon circle mesh (used to decant ethanol from the bulk insect bottle during step 7.9 of the FAVIS protocol) and/or any archive label inside your bulk insect bottle, remove it with a pair of large tweezers and place it inside a large glass petri dish.

Put the petri dish under a microscope. Using the small tweezers, transfer the insects that are stuck to the round mesh and/or the archive label into its respective the bulk insect bottle.

Add the appropriate amount of lysis buffer to each insect bottle according to Table 1 using a graduated cylinder and a small funnel to get the right amount of buffer. The funnel is important so that the lysis buffer is poured slowly and accurately from the 1L bottle into the graduated cylinder, reducing foaming.

A	B	C
Sample weight (g)	Lysis buffer (mL)	Proteinase K (μL)
< 5.00	30	30
5.00 – 9.99	50	50
10.00 - 19.99	100	100
20.00 - 29.99	200	200
30.00 - 39.99	300	300
> 40.00	400	400
Blank sample	50	50

Table 1. Specification on amount of lysis buffer and proteinase K to be added to each insect bottle relative to the insect biomass of each sample.

NOTE: When adding the lysis buffer to each insect bottle, use the buffer to rinse the insect bottle walls so that all insect specimens that are attached to the walls fall at the bottom of the bottle and are soaked in the lysis buffer.

Add the correct amount of proteinase K as shown in Table 1 to each insect bottle.

NOTE: To speed up this step you can first add proteinase K to the lysis buffer before adding the mixed solution to the insect bottles. To minimize waste, first calculate how much buffer you will need for the samples you are processing that day (you will know this after having the wet-weight of each sample). Add the correct amount of proteinase K to the total amount of buffer and then simply add the correct amount of “lysis buffer + proteinase K” solution to each insect bottle according to Table 12.

Gently pour the contents of the insect bulk sample into its respective DT-50 rotor stator tube. Make sure to scoop out any remaining insects from the bottle using the different spatulas. Close the rotor stator tube with the lid.

NOTE: For samples of up to 50 mL lysis buffer, all contents will fit in one rotor stator tube and homogenization can be performed in one go. However for larger samples ( $$≥ 100 mL) you have to perform several rounds of homogenization of 50 mL batches depending on the size of the sample. You can use the same rotor stator tube for homogenizing several 50 mL batches of the same sample. After each homogenization round, transfer the homogenate of that round into an appropriate storage bottle that can accommodate the entire insect soup after all batches from that sample are processed.

Processing one sample at a time, assemble the 50 mL rotor statue tube in the Ultra Turrax Drive and homogenize the insects + lysis buffer solution for 2 minutes at 6000 rpm. Spray the UltraTurrax instrument with 10% bleach after homogenizing each sample.

For the blank, pour 50 mL of the lysis buffer solution into a rotor stator tube and homogenize it as you did for the real samples.

Move the rotor stator tube to its respective working station. Let the sample sit for a while after homogenization for the foam to go down. Then open the rotor stator tube and transfer the homogenate back to the original insect sample bottle. For the blank sample use the 500 mL HDPE bottle to store the homogenized buffer. Use the spatula to transfer all the homogenate that is stuck to the rotor stator tube walls.

Close the rotor stator tube with the lid and discard it.

Proceed with sample pre-incubation in a water bath for 30-40 minutes depending on the biomass of the sample (see NOTE below) followed by incubation at 56°C for 2hr and 45 min in a dry shaking incubator at 90 rpm.

NOTE: It is important to preheat the insect bottles in a water bath before starting the incubation period as they take time to heat up to the desired incubation temperature of 56°C in a dry incubator. The water bath speeds up this process considerably and ensures that each sample is at 56°C when starting the incubation period in the dry incubator. You can use a commercial shaking water bath or you can create your own one as we did in our lab (see details in Favis protocol, step 11: : It is important to preheat the insect bottles in a water bath before starting the incubation period as they take time to heat up to the desired incubation temperature of 56°C in a dry incubator. The water bath speeds up this process considerably and ensures that each sample is at 56°C when starting the incubation period in the dry incubator. You can use a commercial shaking water bath or you can create your own one as we did in our lab (see details in Favis protocol, step 11: https://www.protocols.io/view/favis-fast-and-versatile-protocol-for-metabarcodin-kqdg36261g25/v2) . For small size samples (30 mL, 50 mL, 100 mL) it takes 30 minutes of pre-heating in the water bath for the samples to reach the desired temperature of 56°C, for large samples (200 mL, 300 mL, 400 mL) it will take 40 minutes. These timings are specific to the bottles we use to collect insects and the temperature of the tap water that circulates in the water bath (60°C in our case) and they might need to be adjusted for your own project depending on the bottles and water bath you use.

Remove homogenized insect bottles from the dry incubator and move each bottle back to its respective working station.

We have used this protocol to extract DNA from 870 samples from the Insect Biome Atlas project (www.insectbiomeatlas.org) that had first been subjected to mild lysis following the FAVIS protocol. If your original bulk insect sample has first been processed through the FAVIS protocol as ours, you should combine the insect soup (homogenate) obtained in step 16 above with the lysate obtained from the FAVIS protocol before proceeding with DNA purification to make sure you use all available DNA from each catch.

Thoroughly thaw the corresponding lysate from the mild lysis protocol for each sample.

Pour the lysate into the corresponding homogenate bottle and close with the lid.

NOTE: For samples with ≤200 mL lysate, this can be done in the same bottle but for samples with 300 and 400 mL lysate, a 1000 mL bottle is needed.

Mix the combined homogenate+lysate solution thoroughly by inverting the bottle several times until you obtain a clear mixture.

In the next steps we will transfer a 1.8 mL aliquot of the homogenate to a 2 mL microtube which will be used as a working-stock for DNA purification. If you proceed with DNA purification straight away you can save time by transferring the precise aliquot of homogenate (we use 225 μL) directly from the homogenate bottle into a deep well plate and proceed with DNA purification.

Gather a 1000 μL micropipette, one box of 1000 μL filtered tips and a 96 well microtube rack.

Gently shake the insect soup bottle to homogenize the liquid inside before taking the aliquots.

Cycle through each work station and transfer 2x900 μL aliquots of homogenate from each bottle into its corresponding microtube.

Fill in the microtube rack with the microtubes starting with A1 to A12, B1 to B12, and so on. Leave the last four wells (H9; H10; H11 and H12) empty as these will serve as negative (H9) and positive (H10) DNA purification controls and negative (H11) and positive (H12) library preparation controls. Label each 96-well microtube rack with a unique label.

The homogenate bottles and homogenate aliquots in the microtube racks should be individually labelled with a unique ID and stored at -20°C for long term storage if not used straightaway.

Proceed with DNA purification following step 16 of the FAVIS protocol (https://www.protocols.io/view/favis-fast-and-versatile-protocol-for-metabarcodin-kqdg36261g25/v2). In our project we purified 225 uL of homogenate using the KingFisher^TM Cell and Tissue DNA Kit - THERMO SCIENTIFIC^TM (discontinued) on a KingFisher Flex robot - THERMO SCIENTIFIC^TM (ref. 5400620).