Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

The overall goal of this protocol is to improve investigations of trophic ecology, especially from field samples. This method can help answer key questions in the field of food web ecology, such as the impact of invasive species on trophic interactions. The main advantage is the additional application to more time integrating trophic analyses for the exact same specimens and that it can be applied by virtually every ecologist.

Though we established this approach to provide insight into prey consumption of aquatic consumers, it can also be applied for other purposes, for example, to determine the species composition within benthic macroinvertebrate samples. Demonstrating the procedure will be Christian Sodemann, a technician from our laboratory. To start this experiment, transfer previously dissected predator gastrointestinal tract to a two-milliliter safe-lock reaction tube containing 440 microliters of salt extraction buffer.

Immediately freeze the samples at minus 20 degrees Celsius until DNA extraction. After removing samples from the freezer, start DNA extraction by using forceps to add one five-milliliter stainless steel bead to each sample tube, and then leave the samples on the bench at room temperature to thaw. Using a bead mill, homogenize the samples for one minute at 15 hertz.

After spinning down the samples shortly, add 90 microliters of 10%SDS and five microliters of 10 milligrams per milliliter proteinase K to the samples. Next, thoroughly vortex the samples, and incubate them at 60 degrees Celsius with constant shaking at 400 rpm on a ThermoMixer for one hour. To further promote lysis, vortex the samples every 10 minutes during this incubation.

After a short centrifugation spin, pipette 350 microliters of five molar sodium chloride into the samples. Vortex for 30 seconds to one minute to completely mix the samples. Then, centrifuge at 16, 200 times g at five degrees Celsius for 40 minutes.

So one of the most critical things when taking the samples out of the centrifuge is to avoid that beads move and disturb the pellet containing all the particles that need to be removed by the step. Next, carefully transfer approximately 600 microliters of supernatant into a new 1.5 milliliter tube, making sure not to disturb the pellet. Add 600 microliters of ice cold isopropanol, and carefully invert the tube a few times.

Store at minus 20 degrees Celsius overnight. After taking the samples out of the freezer, centrifuge them at 16, 200 times g at room temperature for 20 minutes. Discard the supernatant while making sure not to lose the pellet, and use a tissue paper to carefully dry the tube.

Wash these DNA-containing pellets by adding 200 microliters of 70%ice cold ethanol and then centrifuging at 16, 200 times g at room temperature for 10 minutes. Discard the supernatant with caution, and use lint-free tissue paper to carefully dry the tube. Finally, using a 10 microliter pipette adjusted to eight microliters, carefully remove the remaining supernatant, making sure not to disturb the pellet.

Dry the pellet until all ethanol is evaporated. Then add 50 to 100 microliters of nuclease-free water, vortex briefly, and leave at four degrees Celsius overnight to dissolve the pellet. To verify functional efficiency of DNA samples extracted here, test each of them by PCR, using universal primer sets suitable for the respective food source.

Start by adding primers, dNTP mix, reaction buffer, Taq DNA polymerase, and purified water in a 1.5 or 2.5 milliliter reaction tube and vortexing the standard reaction mixture. Now, for each DNA sample, transfer one microliter of corresponding DNA extract into nine microliters of the standard reaction mixture within a PCR plate. Close the plate, and place it into the thermocycler and start the PCR program.

After the finished PCR reaction, use a short run option of a mini centrifuge to spin down the PCR products. Then mix three microliters of each PCR product with one microliter of six times loading dye. Load these samples into gel slots of a previously prepared agarose gel.

Load 1.5 microliters of a 100 base pair DNA ladder into one slot of the gel as a size standard. Close the lid of the electrophoresis unit, connect it to a power supply, and run the gel at 100 volts per centimeter for 35 minutes. After the electrophoresis run, remove the gel, and place it into a previously prepared staining bath for approximately 10 minutes.

Finally, remove the gel from the staining bath, and place it onto a gel documentation system, and visualize it according to the manual. To analyze predator gut content, use previously prepared working solutions of DNA extract. Perform separate PCR reactions for each of the 16 prey group-specific primer sets.

Include one positive control with DNA from a specimen belonging to the respective prey group and one negative control with DNA from the predator species. It is crucial to document the allocation of sample within each PCR plate exactly to avoid pooling different samples while loading them on a sequencing plate for the detection of an allotment sequencer and ultimately assigning the wrong results to a consumer individual. Then, run the PCR reactions using the same protocol as previously described, adjusted to the specific annealing temperature and cycle number for each primer set used.

To detect the amplified fragments in these PCR reactions, run automated fragment analyses in two batches, A and B.Prepare a mixture containing sample loading solution, or SLS, and the respective size standard for each batch. Next, thaw the previously frozen PCR products of the eight PCR reactions belonging to the respective batch, making sure they are kept in the dark. Use a short run option of a mini centrifuge to spin them down.

On a sequencing plate, load the number of wells corresponding to the number of samples to be analyzed with 22 microliters of the respective SLS size standard mixture for each sample. Then load one microliter of PCR product of each of the eight PCR reactions into the respective wells of the plate. Next, use a short run option of a mini plate centrifuge to carefully spin down this mixture.

After that, cover each well with one drop of mineral oil. Then fill the reaction wells of a buffer plate with 250 to 300 microliters of DNA separation buffer. Use the capillary sequencer software to create a sample setup according to the manufacturer's instructions.

Make sure that allocation of samples within the sample setup matches the sample allocation in the sequencing plate and that the sample setup contains the method appropriate to process the respective batch. When analyzing results, it is important to visually recheck the electropherogram of each sample and confirm/unconfirm each peak called for each marker primer set separately and insert uncalled peaks that fit the criteria of a marker primer set manually or delete wrong ones. After conducting feeding experiments to determine the effectiveness of group-specific primer sets established for the PCR analyses performed in this protocol, PCR results using DNA from the predator gastrointestinal tract were analyzed using agarose gel electrophoresis as well as single-strand conformation polymorphism, or SSCP.

Agarose gel electrophoresis revealed single bands of double-stranded DNA, which could not be distinguished between the different prey species studied. In contrast to that, SSCP electrophoresis was successful in distinguishing between the different prey species by comparing the gut content samples with the reference samples. DNA extracted from the water mite's gut after feeding experiment was sequenced in different time periods after prey consumption.

From one to 50 hours after feeding, the relative amounts of detected prey DNA was significantly reduced. DNA extracted from the invasive amphipod, Dikerogammarus villosus gut, was tested for macroinvertebrate prey DNA using 16 group-specific primer sets described in this protocol. Only 16%of the D.villosus gut contents was positive for DNA belonging to any of the 16 targeted prey taxa.

Furthermore, DNA of 12 tested prey taxa was found in at least one sample while DNA of four taxa was never detected. While attempting this procedure, it's important to remember to reassure that the primers used are specific for the taxa within your system. After watching this video, you should have a good understanding of how to extend your research on trophic interactions by supplementing the methods routinely used within your lab with genetic analyses.