Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

The aim of this procedure is to demonstrate the methods and techniques commonly used for soil sampling and isolation of entero pathogenic nematodes. This is accomplished by first collecting soil samples by a transect approach, or as demonstrated a random sampling approach. Next, the soil samples abated with insects to selectively recover entero pathogenic nematodes.

The final step is to collect the nematodes from infected insects using a modified white trap. Ultimately, these techniques represent key steps for the successful establishment of entero pathogenic nematode cultures in the laboratory, and form the basis for downstream bioassays. The advantage of the soil sampling technique is that we can go to the field and collecting mature stages of pathogenic nematodes.

I rather than sampling for insects that are infected with this nematodes, which are seldom encounter. The random sample strategy we employ is commonly used to assess the diversity of pathogenic nematodes from a large geographical region or area. This technique was first described by betting in Accur in 1975.

It is a selective procedure based on the premise that free living stages will be attracted to insect hosts and parasitize them. Demonstrating The procedures will be Rue Orozco graduate student, and Ming Mingle research technician in my laboratory.Laboratory. The site for collecting samples should have a minimum area of two square meters, and if possible, be greater than four square meters.

For each soil sample, take at least three smaller subsamples within the DM a C sample area. The subsamples can be combined if it meets the study's objectives. Collect the soil samples as a depth of zero to 15 centimeters.

Each sample should be 250 grams to one kilogram. Use soil CORAs trows, hand shovels or similar tools, and collect from at least five random sites within each selected sampling area. Each sample should be double bagged and labeled with a waterproof marker.

The label should contain information on the site, location, elevation, date, and environmental variables such as habitat description with associated vegetation and temperature at the site. Make a note of the local insects and other invertebrate species. Collect species that can't be immediately identified for later identification in the lab between collection sites thoroughly wash the collecting tools with water, then disinfect them using 70%ethanol or 0.5%bleach.

Store the soil samples in a cooler at eight to 15 degrees Celsius for transportation. CES apart one portion of the sample to analyze the gross characteristics of the soil like its composition, texture, conductivity, and so forth. From each soil sample, remove the debris that could lead to seic microbe contamination.

Then moisten the soil with water so the nematodes therein can easily move about. Load 200 to 250 grams of the moist soil into a plastic container with a lid. Then add insect baits.

Five to 10 insects will suffice. Attach the lid, flip the container over and put it in the dark. At room temperature every two to three days, remove the dead insects and as needed, replace them with healthy insects for additional weighting.

If the insect cadaver is brown or ochre, this suggests Stein and Nema parasitizing parit. Whereas if the cadaver is brick red to dark purple, anticipate hetero. Use a modified white trap to collect the nematodes that have parasitized the insect baits.

First, place the lid of a 50 or 60 millimeter Petri dish into the base of a 100 millimeter dish. Then place a sheet of circular watman. Number one filter paper in the smaller dish.

Rinse the insect cadavers with sterilized water and transfer them to the filter paper. Spread out so that they are not in contact with each other. Fill only the larger dish with about 20 milliliters of distilled water and cover the assembly with the larger dishes.Lid.

Label the dishes with the nematode name, the insect baiting date and date. The trap is set up. Keep the traps at room temperature and monitor for infective, juvenile nematodes or IJs emerging from the insect cadavers.

This can take 10 to 25 days and is dependent on the temperature and moisture level of where the trap is kept. If the water level in the trap has evaporated, be sure to refill it on plates with IJs. Harvest the water from the larger dish, which should contain nematodes.

Allow the nematodes to sink for a few minutes and then slowly decant the water. Once the nematodes remain in a small volume of water, rinse them off a few more times by adding back water and then carefully pouring it off. Then make serial dilutions to estimate the nematode population.

In a 250 milliliter culture flask, adjust the concentration of the nematode to between one and 3000 nematodes per milliliter. Store this suspension between 10 and 20 degrees Celsius. Generally in nature, chances to find insects parasitized by entero pathogenic nematodes are very low.

Thus, the outlined protocol using G melanoma larvae as bait was used to isolate such nematodes at a much more efficient rate. After five days, the insects were infected and had a red coloration. Modified white traps showed nematode trails.

After 15 days from these traps, the nematodes collected in water, had a clean appearance, and were free of insect tissues. A single extraction cycle using gallium Melania larvae may be insufficient for recovering all Oma pathogenic nematodes that are in a soil sample. So we recommend doing this multiple times.

Also, we advise users to consider other insects rather than Galleria, which will also enhance recovery chances. Debating of soil samples with insects is a technique that can be also considered for isolation of other insect pathogens such as bacteria or fungi.