The authors wish to thank past members of the Stock lab: Sam-Kyu Kim, Kathryn Plichta and Victoria Miranda-Thompson for their contributions to the improvement of many of these protocols. We acknowledge the National Science Foundation support (grants IOS-0840932 and IOS-0724978) to S. P. Stock.

1. Soil Sample Collection
<ol>
	<li>Consider covering a minimum area of 2 - 4 m2 for each sampling site.</li>
	<li>Collect soil samples at a depth of at least 15 cm (Figure 4).</li>
	<li>Take at least 5 random samples within this area.</li>
	<li>Take 3 subsamples per sample. Depending on the objective of the study, either combine the subsamples or keep them separate.</li>
	<li>Place each sample in a plastic bag. Consider double-bagging to avoid leaking of samples (Figure 4).</li>
	<li>Label samples with a waterproof marker. Include the following information from each sampling site: Site information (including locality/area/site name and GPS coordinate information), date, habitat, associated vegetation, temperature, elevation, etc. 
	NOTE: If applicable, collect and/or record the presence of insects or other invertebrates collected with the sample for subsequent identification. They may represent potential natural EPN hosts.</li>
	<li>Clean collecting tools between samples by thoroughly washing with water and/or disinfecting them with 70% ethanol or 0.5% bleach solution.</li>
	<li>Keep samples in a cooler (8 - 15 &#176;C) during transport to the laboratory.</li>
	<li>Take a portion of the soil sample for analysis to obtain information on soil composition, texture, moisture, electrical conductivity or other desired soil parameters.</li>
</ol>
2. Nematode Isolation from Soil Samples: Insect-baiting Technique
<ol>
	<li>Remove any debris (i.e., rocks, pieces of wood or bark, leaves, etc.) collected with your samples to avoid contamination with saprobic microorganisms.</li>
	<li>Add water to moisten the soil and facilitate the movement of nematodes. 
	NOTE: Use a spray bottle to slowly increase the moisture level of the soil.</li>
	<li>Place approximately 200 to 250 ml of moist soil in a clean plastic container with a lid.</li>
	<li>Add insect baits. Consider 5 to 10 insects to the soil surface of each sample (Figure 5).</li>
	<li>Cover the container with a lid and turn containers upside down.</li>
	<li>Maintain containers in the dark and at RT (usually 22 - 25 &#176;C).</li>
	<li>Check containers every 2 - 3 days and remove dead insects. 
	NOTE: add additional healthy insects to each container for further baiting of the soil sample.</li>
	<li>Remove dead insects from the containers. NOTE: Cadavers with a brown or ochre coloration are usually parasitized by steinernematids, whereas brick red to dark purple cadavers are parasitized by heterorhabditids.</li>
	<li>Rinse cadavers in sterile water.</li>
	<li>Place cadavers in a modified White trap for recovery of nematode progeny (see procedure below).</li>
</ol>
3. Nematode Recovery from Infected Cadavers: Modified White Trap
<ol>
	<li>Place the top of a 50 (or 60) mm diam. Petri dish inside a larger dish (100 mm).</li>
	<li>Set one single circular filter paper (Whatman #1) inside the smaller dish.</li>
	<li>Place cadavers on the filter paper of the smaller dish making sure cadavers do not touch each other to avoid any contamination.</li>
	<li>Fill the outer (larger) Petri dish with ca. 20 ml of sterile distilled water. Do not add water in the dish that holds the cadavers.</li>
	<li>Cover the large Petri dish and its contents with the lid (Figure 6).</li>
	<li>Label dishes accordingly. Add the following information: Nematode name (species/isolate), infection date (date infection was set) and trap date (this is the date the trap is set up).</li>
	<li>Keep trap at RT until emergence of infective juvenile stages (IJs) occurs. This process can take between 10 - 25 days depending on the nematode species and/or strain considered.</li>
	<li>Harvest water with IJs by removing the larger dish of the trap and pouring water with nematodes into a beaker.</li>
	<li>Allow nematodes to decant to the bottom of the beaker. This process may take a few minutes.</li>
	<li>Pour water carefully, making sure nematodes remain in the bottom of the beaker.</li>
	<li>Rinse nematodes by adding more water and allowing nematodes to decant. This step can be repeated 2 - 3 times until water is clean.</li>
	<li>Place nematode suspension in a tissue culture flask (250 ml). Keep concentration of the suspension to 1,000 - 3,000 nematodes/ml.</li>
	<li>Store flask with nematode suspension in a cold room or in incubator between 10 - 20 &#176;C. 
	NOTE: check stored flasks periodically as shelf life of EPN is variable. Usually steinernematids can be stored for 6 - 12 months without the need of subculturing, whereas heterorhabditids may require more periodic check-ups.</li>
</ol>

<ol>
	<li>Bedding, R. A., Akhurst, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+the+detection+of+insect+parasitic+rhabditid+nematodes+in+soil.">A simple technique for the detection of insect parasitic rhabditid nematodes in soil.</a> Nematologica. 21, 109-110 (1975).</li><li>Boemare, N. E., Akhurst, R. A., Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K. -. H., Stackebrandt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Genera Photorhabdus and Xenorhabdus. In The Prokaryotes. , 473-488 (2007).</li><li>Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Entomopathogenic Nematology. , (2002).</li><li>Gaugler, R., Kaya, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Entomopathogenic Nematode s in Biological Control. , (1990).</li><li>Hazir, S., Keskin, N., Stock, S. P., Kaya, H. K., &#214;zcan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+distribution+of+entomopathogenic+nematodes+(Rhabditida:+Steinernematidae+and+Heterorhabditidae)+in+Turkey.">Diversity and distribution of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Turkey.</a> Biodiversit., &amp; Conservation. 12, 375-386 (2003).</li><li>Kaya, H. K., Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomopathogenic+nematodes.">Entomopathogenic nematodes.</a> Annual Review of Entomology. 38, 181-206 (1993).</li><li>Kaya, H. K., Stock, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques in insect nematology. In Manual of techniques in insect pathology. , 281-324 (1997).</li><li>Lacey, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of techniques in invertebrate pathology. , (2012).</li><li>McCoy, C. W., Shapiro-Ilan, D., Duncan, L., Nguyen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomopathogenic+nematodes+and+other+natural+enemies+as+mortality+factors+for+larvae+of+Diaprepes+abbreviatus+(Coleoptera:+Curculionidae).">Entomopathogenic nematodes and other natural enemies as mortality factors for larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae).</a> Biological Control. 19, 182-190 (2000).</li><li>Meyling, N. V., Eilenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+and+distribution+of+soil+borne+entomopathogenic+fungi+within+a+single+organic+agroecosystem.">Occurrence and distribution of soil borne entomopathogenic fungi within a single organic agroecosystem.</a> Agriculture, ecosystem., &amp; environment. 113, 336-341 (2006).</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=.">.</a> Taxonomy and biology of Steinernematidae and Heterorhabditidae. In: Entomopathogenic Nemtaodes in Biological. , 23-61 (1990).</li><li>Stock, S. P., Gress, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+phylogenetic+relationships+of+entomopathogenic+nematodes+(Steinernematidae+and+Heterorhabditidae)+from+the+Sky+Islands+of+southern+Arizona.">Diversity and phylogenetic relationships of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from the Sky Islands of southern Arizona.</a> Journal of invertebrate pathology. 92, 66-72 (2006).</li><li>Stock, S. P., Hunt, D. J., Grewal, P. S., Ehlers, R. U., Shapiro-Ilan, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nematode+morphology+and+systematics.">Nematode morphology and systematics.</a> Nematodes as biological control agents. CAB International Publishing. , 3-43 (2005).</li><li>Stock, S. P., Goodrich&#8211;Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomopathogenic+nematodes+and+their+bacterial+symbionts:+The+inside+out+of+a+mutualistic+association.">Entomopathogenic nematodes and their bacterial symbionts: The inside out of a mutualistic association.</a> Symbiosis. 46, 65-75 (2008).</li><li>Stuart, R. J., Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patchiness+in+populations+of+entomopathogenic+nematodes.">Patchiness in populations of entomopathogenic nematodes.</a> Journal of Invertebrate Pathology. 64, 39-45 (1994).</li><li>White, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+obtaining+infective+nematode+larvae+from+cultures.">A method for obtaining infective nematode larvae from cultures.</a> Science. 66, 302-303 (1927).</li></ol>

No conflicts of interest declared.

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 regions4, ...

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 Heterorhabditidae11,13. 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 living nematode stage, the third-stage infective juvenile (also known as dauer juvenile, infective juvenile or IJ), which lives in the soil. Once inside the insect, the nematodes release the bacteria into the insect&#8217;s hemolymph, which kill the insect host by massive septicemia. The bacteria also degrade the insect&#8217;s tissues and become the nematodes&#8217; food source, allowing them to mature and multiply. Usually, one or two generations of adult nematodes are produced within the insect cadaver. The progeny of the last adult generation reassociates with a few bacterial cells in a more specialize manner than their previous nutritional relationship, and nurture these cells in their intestines as they move out from the insect carcass into the soil where they will await another insect to parasitize6,11,14 (Figure 1).
Since their discovery in 1927, EPN have been considered valuable alternatives to chemical pesticides as they can parasitize a wide range of insects that are agricultural pests3,4,5. However, over the past two decades, these nematodes and their symbiotic bacteria have been recognized as a malleable model system for studying biological, ecological and evolutionary aspects of host-microbe interactions14.
The goal of this presentation is to show the methods and techniques most commonly used for soil sampling and isolation of EPN. A variety of tools is available and can be used for collecting soil samples including: soil corers, trowels, post-hole diggers, augers, sampling tubes, hand shovels, among others (Figure 2).
Depending on the purpose of the study, two sampling strategies can be considered: a) stratified, b) random sampling (Figure 3). The stratified sampling is usually used as part of an intensive study in a specific or demarked area over a given period of time. In general, a transect is demarked and soil samples are taken at stipulated intervals in the transect over a period of time (Figure 3A)15. The random sampling is generally employed when focusing on an extensive area. This sampling strategy has been used for studying the diversity of EPN from a large geographic area or region (Figure 3B)12. A number of factors can be considered depending on the focus of the study including a diverse range of elevations, soil textures and habitats (e.g., cultivated fields, forests, pastures, parks, seashores, riparian areas, etc.).
We show the insect-baiting technique, which was originally described by Bedding and Akhurst1 and represents a simple and selective alternative for recovering EPN from soil samples. This is a selective procedure that is based on the premise that the nematodes (IJ stage) will be attracted to an insect host and parasitize it. This technique can also be considered for isolation of other insect pathogens such as fungi, and bacteria8,10.
We also demonstrate the modified White trap technique which is used for retrieving nematode progeny from infected insect hosts7,14. This is an effortless method that offers the advantage of retrieving a &#8216;clean&#8217; nematode progeny free of debris from the degrading insect cadavers.
Finally, the techniques described and shown in this article correspond to those conceived and/or performed in our laboratory as well as those described by various colleagues and collaborators.

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

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 Sample Collection
Third-stage infective juveniles of EPN are the only free living stages in these nematodes&#8217; 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 ke...

Watch this Scientific Journal Video about Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae at JoVE.com

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.

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 ...

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24.9K Views.  University of Arizona. 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.

Video: Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

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 ...

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.

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.

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

Bioengineering

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

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 ...

Immunology and Infection

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 ...

Biology

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

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 ...

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

Summary

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Chapters in this video

Design and Operation of a Continuous ¹³C and ¹⁵N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

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

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

Rapid Isolation of Wild Nematodes by Baermann Funnel

Culturing and Genetically Manipulating Entomopathogenic Nematodes

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

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

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