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

1. In vivo Rearing of Entomopathogenic Nematodes with their Symboitic Bacteria
<ol>
	<li>Invert a 100 x 15 mm plastic Petri dish and place two discs of filter paper (90 mm) in the lid of the dish.</li>
	<li>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.</li>
	<li>Add 10 last instar larvae of the greater waxmoth Galleria mellonella to the dish. The goal is to provide approximately 100-200 IJs/larva.</li>
	<li>Cover the lid with the bottom of the Petri dish (Figure 1) and label the Petri dish. Mention the following information: nematode species name (if known); isolate code/designation; date infection trap was set.</li>
	<li>Place the dish inside a loosely sealed plastic bag (to conserve moisture) and keep it in the dark at either room temperature or in an incubator between 20-25 &#176;C. Check dishes daily 
	NOTE: Darkness facilitates nematode infection, as it mimics natural infection conditions in the soil.</li>
	<li>After 3-5 days, remove cadavers with signs of nematode infection to a modified White trap7. 
	NOTE: If the cadavers smell putrid, this may be an indication that the culturing was not successful and/or there was contamination. Set a new infection chamber with a new batch of nematodes and&#160;insects.</li>
</ol>
2. In vitro Rearing of Entomopathogenic Nematodes with their Symbiotic Bacteria
<ol>
	<li>Liver-kidney Agar Method9,19
	<ol>
		<li>Chop liver and kidney into small pieces (2 cm3 or smaller) and place into a blender with NaCl, agar and 300 ml of water and blend until the meat becomes a liquid pulp, thick paste or puree. Then, transfer mixture to a 1 L Erlenmeyer flask.</li>
		<li>Add the final 200 ml of water to the blender vessel and decant any remaining media into the Erlenmeyer flask. Autoclave for 15 min at 121 &#176;C. After autoclaving allow agar to cool to touch before pouring. 
		NOTE: Pipetting of medium is not recommended because this medium tends to have chunks of meat.</li>
		<li>Pour ~20 ml of agar onto 5 (or 6) cm Petri dishes while hand stirring or swirling the Erlenmeyer flask between pours for a more homogenous media. Allow agar to solidify and store dishes at 4 &#176;C until needed 
		NOTE: Use a flame-sterilized metal spatula to break up any large chunks of meat poured into the Petri dish.</li>
	</ol>
	</li>
	<li>Lipid Agar Method9,19
	<ol>
		<li>Prepare lipid agar medium by mixing together nutrient broth, agar and yeast extract and pour mix into a 2 L Erlenmeyer flask. Add distilled H2O and MgCl2&#8226;6H2O and autoclave for 15 min at 121 &#176;C.</li>
		<li>Add sterile mix of corn oil and corn syrup mix and stir or swirl vigorously and often to disperse oil droplets evenly. 
		NOTE: Sterilize corn oil and syrup by placing the needed volume in a UV cross-linker. The oil will not dissolve into the liquid, but make sure it is in tiny droplets.</li>
		<li>Pour ~20 ml agar onto a 5 (or 6) cm Petri dishes and allow agar to solidify and store dishes at 4 &#176;C until needed.</li>
		<li>Streak the desired symbiotic bacteria onto the lipid agar plate and incubate plates at 28 &#176;C for 24-48 hr. 
		NOTE: Spread 100-200 &#181;l of overnight (12-16 hr) LB subculture.</li>
		<li>The day after, add approximately 0.4 ml of surface sterilized nematode suspension (eggs or infective juveniles) and incubate plates at room temperature in the dark or in an incubator at 22 &#177;3 &#176;C. 
		NOTE: Do not add more than 0.4 ml of nematode suspension as it can create an excessively wet environment that is unfavorable to nematode growth.</li>
		<li>Monitor cultures daily. Cultures should produce a new generation of IJs in about 12 days (Figure 2).</li>
		<li>Transfer bottom dish with agar when nematodes are seen crawling the sides of the dish (Figure 3A) to a modified White trap (see Orozco et al.12) to harvest IJs (Figure 3B). Perform this step under a laminar flow hood to reduce contamination of the media.</li>
		<li>Collect IJs from the modified White trap upon emergence and rinse IJ suspension to remove any medium debris. Store IJ in sterilized distilled water at 10 &#176;C (in a cold-temperature incubator or cold room) or use immediately if needed. 
		NOTE: Depending on the goal of the study, seed the plates with either symbiont-colonized or aposymbiotic nematodes.</li>
	</ol>
	</li>
</ol>
3. In vitro Rearing of Aposymbiotic (Symbiont-free) Entomopathogenic Nematodes
<ol>
	<li>Infect 10-20 G. mellonella larvae with IJs of the desired nematode species (see section 1). After 3 or 4 days (at this time IJs should have matured to first generation adults) collect cadavers. 
	NOTE: Time to maturity and number of females needed to obtain an adequate number of eggs will vary by species. Infect more G. mellonella larvae if necessary and start dissections as early as the 48 hr post-infection to assess whether they are in the right developmental stage.</li>
	<li>Place each cadaver individually in a Petri dish with saline solution (M9 buffer18). Dissect a cadaver by pulling its head with a fine tip forceps.</li>
	<li>Collect at least 20 gravid (with eggs inside) females with a fine needle (L-shape preferably) and place them in a watch glass containing 10 ml of M9 buffer (Figure 4A).</li>
	<li>Prepare an axenizing solution by considering the following ingredients: 6.75 ml distilled water, 1.25 ml 5 N NaOH, and 2.25 ml commercially available bleach solution diluted to 3% hypochlorite. 
	NOTE: NaOH is a basic solution&#8212;gloves, goggles and other appropriate protective clothing should be worn. 
	NOTE: Prepare fresh axenizing solution each time, as bleach degrades in light. Prepare several batches of axenizing solution and perform the surface sterilization and harvesting of eggs in multiple watch glasses.</li>
	<li>Rinse females at least three times with M9 buffer filling watch glass with buffer solution and sucking up buffer with a pipette. Make sure the females remain in the watch glass. Repeat this step two more times.</li>
	<li>Immediately add the axenizing solution and allow the females to remain in this solution for no more than 10&#8211;15 min. Observe the nematode tissues beginning to disintegrate while the eggs (which are resistant to the axenizing solution) remain intact.</li>
	<li>Manually disrupt female bodies to speed up the process, by cutting them with a needle or a scalpel (Figure 4B, C). Remove eggs by pipetting them into 1.5 ml microcentrifuge tubes, avoiding adding any undissolved tissue. 
	NOTE: Use as many microcentrifuge tubes as necessary to gather eggs and work quickly, as the viability of the eggs will decrease over a prolonged period of exposure to axenizing solution.</li>
	<li>Vortex tubes for 15 sec using the &#8220;high speed setting&#8221; to further remove nematode tissues attached to the eggs. Alternatively, if a vortex is unavailable, pipet up and down the solution several times.</li>
	<li>Pellet eggs by centrifugation at approximately 18,000 g for 2 min. Increase speed if eggs do not sufficiently pellet. Remove axenizing solution and rinse twice by filling the microcentrifuge tube with sterile distilled water and centrifuge one more time at 900 x g for 1 min.</li>
	<li>After the last wash, resuspend eggs in 300&#8211;500 &#181;l of sterile distilled water and place them in a sterile 3 cm Petri dish or sterilized watch glass. Remember that eggs are now surface-sterilized, so use sterile pipettes and/or pippetter tips and water to maintain cleanness of the eggs.</li>
	<li>Examine eggs under a dissecting microscope (30-50X magnification) to confirm they are intact (Figure 4D) and transfer them to a 5 cm plate with liver-kidney agar (see section 2.1) 
	NOTE: Do not add more than 400 &#181;l of the egg suspension on the plates as it will make the agar excessively wet.</li>
	<li>Label plates with appropriate information and store them upright in the dark at 28 &#176;C. Hatching of eggs should be evident overnight. 
	NOTE: Water may condense on the lid of the dish but it will evaporate after 1 or 2 days. At this time, place the dish in plastic bag to retain moisture of the agar and avoid its drying.</li>
	<li>Observe dishes periodically and discard any dishes that show signs of fungal or bacterial contamination. Once IJs are seen migrating up the wall of the Petri dish, remove the lid and transfer the bottom dish with agar to a modified White trap12. Consider sterile conditions when transferring the dish into the modified White trap to avoid contamination of the exposed media.</li>
	<li>Continue monitoring the White Traps and harvest IJ as needed. 
	NOTE: Aposymbiotic nematodes will continue to migrate into the water of the White trap for many days and even weeks.</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+sampling+and+isolation+of+entomopathogenic+nematodes+(Steinernematidae,+Heterorhabditidae).">Soil sampling and isolation of entomopathogenic nematodes (Steinernematidae, Heterorhabditidae).</a> J. Vis. Exp. (89), (2014).</li><li>Poinar, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+Achromobacter+nematophilus+in+the+infective+stage+of+a+Neoaplectana+sp+(Steinernematidae:+Nematoda).">The presence of Achromobacter nematophilus in the infective stage of a Neoaplectana sp (Steinernematidae: Nematoda).</a> Nematologica. 12, 105-108 (1966).</li><li>Poinar, G. O., Thomas, G. 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Microbiol. 70, 6473-6480 (2004).</li><li>Sicard, M., Hinsinger, J., LeBrun, N., Pag&#232;s, S., Boemare, N., Moulia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecific+competition+between+entomopathogenic+nematodes+(Steinernema)+is+modified+by+their+bacterial+symbionts+(Xenorhabdus).">Interspecific competition between entomopathogenic nematodes (Steinernema) is modified by their bacterial symbionts (Xenorhabdus).</a> BMC Evolutionary Biology. 6, 68-78 (2006).</li><li>Snyder, H., Stock, S. P., Kim, S. 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No conflicts of interest declared.

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...

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.&#160;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.

<table><tbody><tr><td colspan="4">For &lt;em&gt;In vivo&lt;/em&gt; Infections</td></tr><tr><td>100 x 15 Petri dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>Filter paper, 9 cm grade 1 cellulose</td><td>Whatman/VWR</td><td>28450-081</td><td>Grade 1 filter paper recommended</td></tr><tr><td>Insect hosts</td><td>Timberline</td><td>http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td><td>&lt;em&gt;Galleria mellonella&lt;/em&gt; are recommended</td></tr><tr><td colspan="4">For Liver-kidney Agar (for 500 ml)</td></tr><tr><td>60 x 15 mm Petri dishes</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>Beef liver, 50 g</td><td>Locally sourced; butcher or supermarket</td><td></td><td>Remaining may be stored frozen and thawed for future use</td></tr><tr><td>Beef kidney, 50 g</td><td>Locally sourced; butcher or supermarket</td><td></td><td>Remaining may be stored frozen and thawed for future use</td></tr><tr><td>Sodium chloride 2.5 g (0.5% final concentration)</td><td>Acros/VWR</td><td>200002-434</td><td>any research grade NaCl can be used</td></tr><tr><td>Agar, 7.5 g (1.5% agar, final concentration)</td><td>HiMedia/VWR</td><td>95026-642</td><td>any media grade agar can be used</td></tr><tr><td>500 ml distilled H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td colspan="4">For Lipid Agar (for 1 L)</td></tr><tr><td>100 x 15 Petri dishes</td><td>VWR</td><td>25384-088</td><td></td></tr><tr><td>Nutrient broth, 8 g</td><td>BD/VWR</td><td>90002-660</td><td></td></tr><tr><td>Yeast extract, 5 g</td><td>EMD/VWR</td><td>EM1.03753.0500</td><td></td></tr><tr><td>Magnesium chloride hexahydrate 10 ml (0.2 g/ml)</td><td>EMD/VWR</td><td>EM-MX0045-1</td><td></td></tr><tr><td>Corn oil, 4 ml</td><td>Any brand</td><td>Locally source; supermarket</td><td>Any brand of corn oil can be used</td></tr><tr><td>Corn syrup, 96 ml combine 7 ml corn syrup in 89 ml heated H&lt;sub&gt;2&lt;/sub&gt;O and swirl for homogeneity</td><td>Karo</td><td>Locally sourced; supermarket</td><td></td></tr><tr><td>Agar, 15 g</td><td>HiMedia/VWR</td><td>95026-642</td><td>any media grade agar can be used</td></tr><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O, 890 ml</td><td></td><td></td><td></td></tr></tbody></table>

in vivo and in vitro rearing of entomopathogenic nematodes (steinernematidae and heterorhabditidae)

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&#8211;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&#8217;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.

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&#8217; 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 t...

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

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.

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

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

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20.5K Views.  University of Arizona. 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.

Video: In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

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

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.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

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.

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

Environment

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field.

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild ...

Genetics

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Biology

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Immunology and Infection

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Optimizing Pinewood Nematode Infection in Pinus pinaster

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily contributed to pine deforestation in Asian countries, e.g., Japan, China, and Korea. Over the last two decades, in Europe, Portugal and Spain have been greatly affected. Research on the mechanisms of PWN infection and/or PWD progression in susceptible host species relies on the controlled infection of pine seedlings under greenhouse conditions. This technique is laborious and mobilizes substantial economic and human resources. Additionally, it can be prone to variability that results from the genetic diversity associated with some pine species but also from the interference of external factors. As an alternative, in vitro co-cultures of pine with PWNs offer a more advantageous system for studying biochemical changes since they a) allow controlling single environmental or nutritional variables, b) occupy less space, c) require less time to obtain, and d) are&#160;free from contamination or from host genetic variation. The following protocol details the standard in vivo PWN infection of Pinus pinaster, the maritime pine, and the establishment of the novel in vitro co-cultures of pine shoots with the PWN as an improved methodology to study this phytoparasite influence on pine volatiles. PWN-induced volatiles are extracted from in vivo and in vitro infected pines by hydrodistillation and distillation-extraction, and the emitted volatiles are captured by solid phase microextraction (SPME), using fiber or packed column techniques.

Author Spotlight: In Vitro Co-Culture System of Pine Shoots and Pinewood Nematode for Studying Host Volatile Response

The protocol describes the in vivo and in vitro pinewood nematode infection of Pinus pinaster and their volatilome analysis through Gas Chromatography (GC) and GC coupled to Mass Spectrometry (GC-MS).

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily ...

In vivo and In vitro Infection of Potato Roots with Plant Parasitic Nematodes for the Assessment of Induced Structural Changes

Soil-dwelling plant parasitic nematodes (PPNs) are important potato pests that cause lesions and/or change plant roots structure, leading to reduced crop fitness and productivity. Research on the cellular and subcellular mechanisms of PPNs infection and development can resort to field plants or seedlings under greenhouse conditions. Field studies are more representative of natural environments but are subjected to the unpredictability of environmental conditions that can heavily influence research outcomes. Greenhouse studies allow higher control over environmental variables and higher safety against contaminants or pathogens. However, in some hosts, genetic diversity becomes an important factor of variability and influences the host-parasite complex response. We have developed in vitro co-cultures of transgenic roots with PPNs as a reliable alternative that occupies less space, requires less time to obtain, and is free from contamination or from host genetic variability. Co-cultures are obtained by introducing aseptic PPNs to host in vitro transgenic roots. They can be maintained indefinitely, which makes them excellent support for keeping collections of reference PPNs. In the present work, a protocol is detailed for the controlled infection of in vivo potato roots with the root lesion nematode and for establishing in vitro co-cultures of potato transgenic roots with the root-knot nematode. The in vitro co-cultures provided a laboratory proxy for the natural potato infection condition and produced nematode life stages irrespective of season or climate conditions. Additionally, the methodology used for structural analysis is detailed using histochemistry and optical microscopy. The acid fuchsin dye is used to follow nematode attack sites on roots, while differential staining with Periodic acid-Schiff (PAS) and toluidine blue O highlights nematode structures in potato internal root tissue.

The protocol describes infection of Solanum tuberosum roots with plant parasitic nematodes under in vivo greenhouse conditions and potato in vitro transgenic roots for histochemical analysis of root structure through optical microscopy.

Soil-dwelling plant parasitic nematodes (PPNs) are important potato pests that cause lesions and/or change plant roots structure, leading to reduced crop ...

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

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

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Rapid Isolation of Wild Nematodes by Baermann Funnel

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

In vivo and In vitro Infection of Potato Roots with Plant Parasitic Nematodes for the Assessment of Induced Structural Changes