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

Summary

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

Abstract

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.

Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Introduction

Many nematodes are associated with insects, and their host interactions range from beneficial to detrimental. Herein, we focus on a group of nematodes that are pathogens of insects, also known as entomopathogenic nematodes (or EPN). Two nematode families belong to this group of nematodes: Steinernematidae and Heterorhabditidae^11,13. Their pathogenic effect is in fact conferred by their interaction with facultative anaerobic enteric bacteria (Xenorhabdus bacteria associate with steinernematids and Photorhabdus bacteria associate with heterorhabditids)². The bacteria are vectored from one insect host to another by the only free li....

Protocol

1. Soil Sample Collection

1. Consider covering a minimum area of 2 - 4 m² for each sampling site.
2. Collect soil samples at a depth of at least 15 cm (Figure 4).
3. Take at least 5 random samples within this area.
4. Take 3 subsamples per sample. Depending on the objective of the study, either combine the subsamples or keep them separate.
5. Place each sample in a plastic bag. Consider double-bagging to avoid leaking of samples (Figure 4).
6. Label samples with a waterproof marker. Include the following information from each sampling site: Site information (including locality/are....

Results

Soil Sample Collection

Third-stage infective juveniles of EPN are the only free living stages in these nematodes’ life cycle which reside in the soil (Figure 1). Therefore, collecting soil samples is a very efficient method to recover of these nematodes. Figure 4 shows a soil profile indicating the depth at which samples should be taken. Preserving the moisture of a sample is a key factor for survival of the nematodes. Consequently, it is important to keep soi.......

Discussion

This demonstration is the first of two presentations that describe a number of techniques that are commonly used for studying EPN and their symbiotic bacteria. These techniques represent key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that employ EPN as model organisms for research.

Various soil surveys of EPN have shown that their abundance and distribution can vary across seasons, habitats and geographic regions^{4, .......}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Galleria mellonella	Timberline	http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG	For insect baiting technique
Filter paper, 55 mm Grade 1 Cellulose	Whatman/VWR	28450-048	Infection chamber and White trap assembly
60 x 15 mm Petri dishes	VWR	25384-092	Infection chamber and White trap assembly
100 x 15 mm Petri dishes	VWR	25384-088	Infection chamber and White trap assembly
ZIPloc plastic bags	Ace Hardware or local hardware store		Soil sampling
Mechanical pipettor P1000, P200 ml	VWR	89130-562; 89130-566	Infection chamber and White trap assembly
Pippetor tips 1000ml, 200 ml	VWR	16466-004 ; 53510	Infection chamber and White trap assembly
Bleach	Ace Hardware or local hardware store		Soil sampling , disinfecting
Sodium hypochlorite	Ace Hardware or local hardware store		Soil sampling , disinfecting
70% Ethyl alochol	AAPER	17212945	Soil sampling , disinfecting
Shovels	Ace Hardware or local hardware store	N/A	Soil sampling
Tubular soil sampler	Accuproducts		Soil sampling
Soil probe, long handle	Nupla	PRB4T 69401 Classic	Soil sampling
Measuring tape	Ace Hardware or local hardware store		Soil sampling
Flagging tape, various colors	Ace Hardware or local hardware store		Soil sampling
Tissue culture flasks	VWR	Falcon, 29185-304	White trap, collection of EPN
Wash bottles, polyethylene, wide mouth	VWR	16650-107	White trap, collection of EPN