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.
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.
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 relationships2-4,6,10,19. 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 3rd stage infective juvenile8,10,13. The bacterium-nematode pair is pathogenic for a wide range of insects and has successfully been implemented in biological control and integrated pest management programs worldwide6,8.
Herein we show a selection of in vivo and in vitro techniques that are frequently exercised for the rearing of EPN under laboratory conditions. In vivo methods contemplate an insect host for the rearing of the nematodes. Usually, immature stages of various insect orders (i.e. Lepidoptera, Coleoptera, Diptera, etc.) are considered suitable hosts. In vivo methods are usually considered for maintenance of nematode cultures in the lab. This method may not be suitable when considering mass production of the nematodes. Large quantities of insect hosts may be required for this purpose demanding more time and additional costs related to the insect rearing.
Entomopathogenic nematodes can also be cultured in vitro on several media. Depending on the goal of the study; in vitro methods may or consider the incorporation of the symbiotic bacteria in the media. In this presentation, we describe two commonly used methods for the propagation of EPN. The ingredients of the media provide a source of nutrients for the symbiotic bacterium and a sterol source for the nematodes. In vitro methods offer the advantage the rearing of EPN without an insect host.
Originally, many of the in vitro media developed were used for the multiplication of EPN when suitable insect hosts are not available. However, over the past years, in vitro rearing methods have become widely employed in research aiming to understand the mutualistic relationship between EPN and their symbiotic bacteria17,19.
The techniques detailed in this presentation correspond to those described by various authors and refined by the Stock 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.
1. In vivo Rearing of Entomopathogenic Nematodes with their Symboitic Bacteria
2. In vitro Rearing of Entomopathogenic Nematodes with their Symbiotic Bacteria
3. In vitro Rearing of Aposymbiotic (Symbiont-free) Entomopathogenic Nematodes
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 rearing of aposymbiotic (symbiont-free) nematodes. Figure 2 shows a lipid agar plate with different nematode stages. Lipid agar plates may also be considered in cross-hybridization tests which are required to validate nematode identity based on the biological species concept. The liver kidney agar method was originally described by Poinar & Thomas (1966)14. This method allows the nematodes to mature and reproduce without an insect host and can be used to rear nematodes without their symbiotic bacteria (i.e. produce aposymbiotic nematodes). A disadvantage of this method is that it is prone to contamination because of their rich nature, allowing unwanted bacteria or fungi to grow. Figure 3A shows liver-kidney agar plates with IJS crawling on the side of the plate. At this stage the bottom portion of the Petri dish can be transferred to a modified White trap. Figure 3B demonstrates a liver-kidney plate with Steinernema nematodes in a modified White trap for the harvesting of IJs progeny. Figure 4A shows a watch glass with gravid Steinernema females prior to their axenization. Figure 4B displays the disruption of the females with a dissecting needle. Figure 4C shows disrupted females in the axenizing solution. Figure 4D shows an intact egg of the nematode Steinernema carpocapsae.
Figure 1. Set up of an infection chamber for in vivo rearing of EPN. Top row shows a 5 cm Petri dish and filter paper. The bottom row displays a 10 cm Petri dish and filter paper. Notice number of insect larvae added to each infection chamber.
Figure 2. Lipid agar method for in vitro rearing of EPN. The image shows a close-up (20X magnification) of a dish with adult nematodes developing on the medium.
Figure 3. Liver-kidney agar plate. Image A on the left shows a dish with successful growth of nematodes. Notice nematodes crawling on the side of the dish. Image B shows a liver-kidney agar plate placed in a modified White trap for the harvesting of IJ nematodes.
Figure 4. Watch glass with gravid females in axenizing solution. Image A shows gravid females in the axenizing solution. Image B shows the grinding of the females with a dissecting needle. Image C displays disrupted females. Image D shows an intact egg of the nematode Steinernema carpocapsae.
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 S. scarabaei are not very virulent to lepidopteran larvae, and need coleopteran larvae such as scarab beetle larvae (Scarabaeidae) for successful rearing in the lab. Another species, S. scapterisci, prefers orthopteran insects such as crickets or mole crickets.
When a suitable host is not available or when experimental conditions require it, in vitro methods can be employed for the rearing of EPN. These methods use media that offer a rich source of nutrients for nematodes and their bacterial symbionts. In this presentation we described two types of agar (liver-kidney and lipid) that can be utilized for the successful growth and reproduction of EPN with or without their symbiotic bacteria.
Users should be aware that the liver-kidney agar is a rich media and is therefore easily contaminated. Sterile technique is recommended in handling both the axenized eggs and the inoculated plates during all steps of the procedure. Plates that are contaminated with bacteria or fungus should be discarded immediately.
When rearing aposymbiotic Steinernema IJs, care should be taken to properly lyse the female nematode tissues, as they could harbor Xenorhabdus bacteria. Also, to validate that IJ are indeed free of symbiotic bacteria, we recommend the grinding of a sample of freshly harvested IJs in LB with a hand-held motor-driven pestle as per (Heungens et al. 2002)7 and plate the suspension onto NBTA media1. If bacteria colonies are found (after overnight incubation) it will be either an indication of a poor axenization technique or the result of contamination.
The rearing of aposymbiotic Steinernema nematodes is a technique that can also be considered in a wide array of procedures and experiments. Readers should be aware that the impact of rearing Steinernema nematodes without symbionts over multiple generations may have an impact on nematode fitness and survival over generation times. A few studies suggest the necessity of their pairing in natural systems5,11,15-17. However, these studies have been done on a limited number of species and further exploration is needed.
The authors have nothing to disclose.
The authors wish to thank past members of the Stock lab: Ming-Min Lee, Kathryn Plichta, Victoria Miranda-Thompson and Sam-Kyu Kim for their contributions to the improvement of many of these protocols. This work was funded in part by the National Science Foundation grant IOS-0840932 and IOS-0724978 to S. P. Stock
Material | 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 2.5g ( 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 H20 | |||
For Lipid Agar (for 1 L) | |||
100 x 15 Petri Dishes | VWR | 25384-088 | |
Nutrient Broth, 8 g | BD/VWR | 90002-660 | |
Yeast Extract, 5g | EMD/VWR | EM1.03753.0500 | |
Magnesium Chloride Hexahydrate, 10 ml (0.2g/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 H20 and swirl for homogeneity | Karo | Locally sourced; supermarket | |
Agar, 15 g | HiMedia/VWR | 95026-642 | any media grade agar can be used |
Distilled H20,890 ml |