In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

Summary

The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media.

Abstract

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont–free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock’s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.

Introduction

Entomopathogenic nematodes (EPN) Steinernema and Heterorhabditis spp. (Steinernematidae, Heterorhabditidae) and their bacterial symbionts, Xenorhabdus and Photorhabdus spp (Enterobacteriaceae) are considered an emergent model of terrestrial animal-microbe symbiotic relationships^2-4,6,10,19. Xenorhabdus and Photorhabdus spp. are harbored as symbionts in the intestine of the only free-living stage of the nematodes, also known as the infective juvenile (IJ) or 3^rd stage infective juvenile^8,10,13. The bacterium-nematode pair is pathogenic for a wide range of insects and has successfully....

Protocol

1. In vivo Rearing of Entomopathogenic Nematodes with their Symboitic Bacteria

1. Invert a 100 x 15 mm plastic Petri dish and place two discs of filter paper (90 mm) in the lid of the dish.
2. Evenly distribute 1 ml of the IJ (infective juveniles) water suspension (at a concentration of 1,000-2,000 IJ/ml) on the filter paper.
   NOTE: IJs do not need to be surface-sterilized.
3. Add 10 last instar larvae of the greater waxmoth Galleria mellonella to the dish. The goal is to provide approximately 100-200 IJs/larva.
4. Cover the lid with the bottom of the Petri dish (Figure 1) and label the Petri dish.....

Results

The in vivo rearing method uses live insects as hosts for nematode growth and reproduction. Infection chambers are an efficient method for exposing insects IJs. This is the only stage in the nematodes’ life cycle that vectors the bacterial symbionts from one insect host to another. Figure 1 shows the set up for an infection chamber as well as the materials needed to build this chamber. In vitro rearing methods allow EPN to grow without an insect host but are also implemented for the rea.......

Discussion

Using a suitable host is a key factor for the successful in vivo rearing of EPN. Usually, both steinernematids and heterorhabditids can reproduce and successfully complete their life cycle in larvae of the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae). However, other insect species from different families and/or orders can be considered. A few of the currently described nematode species are known to have specificity for a particular insect host. For example, S. kushidai and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
For In vivo Infections
100 x 15 Petri dishes	VWR	25384-088
Filter paper, 9 cm grade 1 cellulose	Whatman/VWR	28450-081	Grade 1 filter paper recommended
Insect hosts	Timberline	http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG	Galleria mellonella are recommended
For Liver-kidney Agar (for 500 ml)
60 x 15 mm Petri dishes	VWR	25384-092
Beef liver, 50 g	Locally sourced; butcher or supermarket		Remaining may be stored frozen and thawed for future use
Beef kidney, 50 g	Locally sourced; butcher or supermarket		Remaining may be stored frozen and thawed for future use
Sodium chloride 2.5 g (0.5% final concentration)	Acros/VWR	200002-434	any research grade NaCl can be used
Agar, 7.5 g (1.5% agar, final concentration)	HiMedia/VWR	95026-642	any media grade agar can be used
500 ml distilled H2O
For Lipid Agar (for 1 L)
100 x 15 Petri dishes	VWR	25384-088
Nutrient broth, 8 g	BD/VWR	90002-660
Yeast extract, 5 g	EMD/VWR	EM1.03753.0500
Magnesium chloride hexahydrate 10 ml (0.2 g/ml)	EMD/VWR	EM-MX0045-1
Corn oil, 4 ml	Any brand	Locally source; supermarket	Any brand of corn oil can be used
Corn syrup, 96 ml combine 7 ml corn syrup in 89 ml heated H2O and swirl for homogeneity	Karo	Locally sourced; supermarket
Agar, 15 g	HiMedia/VWR	95026-642	any media grade agar can be used
Distilled H2O, 890 ml