This protocol can be used to streamline the isolation and identification of Caenorhabditis nematodes from nature and record important ecological and environmental data associated with the nematodes'natural environment. The main advantages to this technique are its scalability and accuracy, which are made possible by leveraging mobile data collection platforms, cloud-based databases, and standardized data processing functions. To begin, identify a location to survey Caenorhabditis nematodes.
To create a Fulcrum project, create an account with Fulcrum online using a no cost educational agreement and add the nematode field sampling application. Then add the nematode isolation application. Print a set of QR code labels as C-Labels to track the collections and S-Labels to track the nematode isolations.
Attach the C-Labels to Ziploc plastic bags for packing. Keep the set of S-Labels for use in the laboratory. Select the nematode field sampling from the dropdown menu of the Fulcrum mobile app and press plus to start a new record in the project.
Take a photo of the substrate. Tap on the C-Label field and choose scan. Scan the barcode on the collection bag using the mobile device camera, then tap done.
Tap on the substrate field and select a substrate type. Measure the surface temperature of the substrate using the non-contact thermometer and record the value in the substrate temperature field. Also measure and record the ambient temperature and humidity.
Save and record in Fulcrum by tapping on save in the upper left of the screen. Collect a tablespoon of the substrate without sticks or other hard pieces by inverting the collection bag as a glove to pick up the substrate, then seal the bag. For each Ziploc bag, note the C-Label on the bag and attach a matching C-Label to the lid of a 10 centimeter plate spotted with OP50 bacteria.
Transfer one tablespoon of sample from each collection bag to the C-plates using a clean plastic spoon. On the Fulcrum application, choose nematode isolation from the application menu. Then make a new isolation record by tapping the plus icon in the lower right.
Tap on the select button to find the C-Label associated with a sample. Tap on the search icon. Then tap on the scan icon to scan the C-Label QR code.
Look for nematodes on the C-plate with a dissecting microscope. Tap on the worms on sample field to record the presence of nematodes on the sample. If no nematodes are present, parafilm the C-plate and dispose of it in a biohazard bin.
If nematodes are present, isolate up to five nematodes from the C-plate and transfer each to its own S-plate. Next, tap on the S-labeled plates field to enter the S-plate used for this isolation. Tap on the plus in the lower right.
Tap on S-Label and then click on scan to open the device camera, then scan the S-Label QR code on the S-plate. Tap on the save button on the upper right once the isolation record has all information added correctly, then parafilm the-S plates with isolated nematodes and set them aside in an area designated to hold S-plates with nematodes. Sign into the Fulcrum website and select the nematode isolation application.
Click on exporter to download a zip file that contains the nematode isolation s_labeled_plates. csv file. Navigate to the wild isolate genotyping template Google Sheet and copy the Google Sheet.
Place the S-Labels copied from the nematode isolation s_labeled_plates. csv column into the genotyping sheet. Check for proliferating animals on S-plates 48 hours after isolation.
If an S-plate has proliferating animals, enter one in the proliferation 48 column in the genotyping sheet, then move the S-plate to a box labeled 48 hours proliferation box one. Check the S-plates that were not proliferating at 48 hours post-isolation again at 168 hours post-isolation. If an S-plate is now proliferating, enter one in the proliferation 168 column on the genotyping sheet and then move the S-plate to a box labeled 168 hours proliferation box one.
Filter the genotype in Google Sheet to include just the S-Labels in one proliferation box to be lysed, then select the columns S-Label through lysis nodes. Print a lysis worksheet for each proliferation box to be lysed. Prepare 12 well strip tubes for the samples to be lysed.
Uncap one strip tube and add eight microliters of lysis buffer to each cap with a repeat pipetter. Pick three to five animals from the source plates into the cap positions indicated on the lysis worksheet. Repeat for up to eight strip tubes and place the strip tubes in the minus 80 degree Celsius freezer.
Remove the sets of strip tubes and run the lysis program in the thermocycler. Then store the lysates at minus 80 degrees Celsius for up to one week. Remove the strip tubes from the minus 80 degree Celsius freezer and thaw the lysis material on ice.
While the lysis material is thawing, prepare ITS2 and SSU master mixes in separate tubes on ice. Add two microliters of lysate to the appropriate well in the PCR plate with a low-volume multi-channel pipette. Run the PCRs with the appropriate thermocycler program and record which S-Labels yield ITS2 or SSU PCR products in the genotyping sheet.
For each sample that is ITS2 positive, use the remaining ITS2 PCR product for Sanger sequencing and obtain the seq output files for each S-Label from the sequencing platform. Navigate to the NBCI BLAST website in a web browser to begin the BLAST search. For S-plate sequences that BLAST to a Caenorhabditis species, enter the full genus and species name of the top BLAST hit in the species ID column.
Name the strains with unique names, following Caenorhabditis nomenclature conventions and enter the strain names in the strain name column. To process the data, navigate to the Fulcrum website and export the raw project data from the Fulcrum database. Open a new R script and follow the directions in the easyfulcrum vignette to process the collection data.
In NBCI BLAST, the results for S-Label S-05554 show that the top hit is Caenorhabditis briggsae. Additionally, some isolated nematodes require additional effort to genotype. For example, approximately 2%of isolates fail to amplify with the SSU PCR primer set after the first lysis attempt and must be relysed to ensure that the lysis material is suitable for amplification with the ITS2 primer set.
Following this procedure, researchers can explore the ecological and environmental data associated with wild nematode isolates to identify locations and substrate characteristics that are most likely to harbor specific nematode species.
