Name: culturing and genetically manipulating entomopathogenic nematodes
Uploaded: 2022-03-31T15:00:00.000+00:00
Duration: 7 min 17 s
Description: Watch this Scientific Journal Video about Culturing and Genetically Manipulating Entomopathogenic Nematodes at JoVE.com

We thank members of the Department of Biological Sciences at George Washington University for critical reading of the manuscript. All graphical figures were made using BioRender. Research in the I. E., J. H., and D. O&#39;H. laboratories have been supported by George Washington University and Columbian College of Arts and Sciences facilitating funds and Cross-Disciplinary Research Funds.

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K., Kaya, H. K., Goodrich-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=Comparative+study+of+the+entomopathogenic+nematode,+Steinernema+carpocapsae,+reared+on+mutant+and+wild-type+Xenorhabdus+nematophila.">Comparative study of the entomopathogenic nematode, Steinernema carpocapsae, reared on mutant and wild-type Xenorhabdus nematophila.</a> Biological Control. 29 (3), 382-391 (2004).</li><li>McMullen, J. G., 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=In+vivo+and+in+vitro+rearing+of+entomopathogenic+nematodes+(Steinernematidae+and+Heterorhabditidae).">In vivo and in vitro rearing of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae).</a> Journal of Visualized Experiments. (91), e52096 (2014).</li></ol>

The authors declare no competing interests.

Understanding the molecular basis of entomopathogenic nematode infection and insect anti-nematode immunity requires the separation of the parasites from the mutualistically associated bacteria13,15,16. The entomopathogenic nematodes H. bacteriophora and S. carpocapsae live together with the Gram-negative bacteria P. luminescens and X. nematophila, respectively17. Both bacterial species have been shown previously to encode factors that confer pathogenicity by targeting insect tissues and counteracting the innate immune system during infection18,19. This hinders efforts to identify the strategies that nematodes have evolved to interact with the insect host. Therefore, identification and functional characterization through gene knockdown of the nematode components that also participate in the infection process can be facilitated by the generation of axenic worms. Here, efficient protocols for producing axenic entomopathogenic nematodes and performing RNAi gene silencing in H. bacteriophora are presented. 
A critical step in both protocols for successfully generating axenic nematodes is the surface-sterilization of H. bacteriophora and S. carpocapsae IJs14,20. This part of the method involves treatment of the worms with bleach solution and is considered crucial for the success of the axenization process because it removes the P. luminescens and X. nematophila bacteria from the nematode cuticle. This is an important procedure to ensure that only the mutualistic bacteria on the surface of the worms are cleared and not those inside the parasites. Completion of this step requires attention because extended bleach treatment will impact nematode survival. 
A similarity of the present axenization protocol for S. carpocapsae nematodes with a previously established method is the use of the X. nematophila &#916;rpoS mutant bacteria which are not able to colonize the worms21. A difference between the current methodology and a previously reported protocol, which introduced surface sterilized nematode eggs onto nutrient agar22, is the addition of antibiotics into the culture media to inhibit microbial contamination that would likely affect nematode growth and fitness. 
RNAi by microinjection is an easy and reliable method of delivering the RNA to the oocytes. The burst of liquid inside the gonad provides visual confirmation of the release of the nucleic acid solution during the injection process. It was shown that RNAi by microinjection is significantly better than RNAi by soaking. This is probably due to the ability of the RNAi solution to reach oocytes that are at different stages of differentiation inside the gonad. 
While optimizing the process, it is recommended to test different concentrations of RNA solution for better results. Various concentrations of RNA ranging from 50 ng/&#181;L to 10 &#181;g/&#181;L were tested. It was found that a concentration of 6 &#181;g/&#181;L worked better than other concentrations. To increase the RNA yield during the in vitro transcription step, the protocol was modified from a 4 hour incubation period to 16 hours. This resulted in a substantial increase in the final yield of RNA. One of the key factors for successful microinjection is the amount of time that a nematode spends on the injection pad. Less time results in a healthy surviving nematode and healthy progeny with an increased chance of observing the intended phenotype.
The current protocol for culturing and genetically manipulating entomopathogenic nematodes is a significant contribution for future studies in the fields of nematology, immunology, and host-parasite interactions. Combining approaches that allow the manipulation of entomopathogenic nematodes will lead to the discovery of the genetic factors that define the interplay between nematode effector molecules and host immune signaling components that encode factors with anti-nematode properties. It will further determine which key nematode molecular components determine the symbiotic relationship with the related bacteria. Answering these questions is important for improving agricultural practices and developing novel means for the control of human parasitic nematodes.

Research on entomopathogenic nematodes (EPN) has intensified over the past few years due primarily to the utility of these parasites in integrated pest management strategies and their involvement in basic biomedical research1,2. Recent studies have established EPN as model organisms in which to examine the nematode genetic components that are activated during the different stages of the infection process. This information provides critical clues on the nature and number of molecules secreted by the parasites to alter host physiology and destabilize the insect innate immune response3,4. Simultaneously, this knowledge is commonly supplemented by novel details on the type of insect host immune signaling pathways and the functions they regulate to restrict the entry and spread of the pathogens5,6. Understanding these processes is crucial for envisioning both sides of the dynamic interplay between EPN and their insect hosts. Better appreciation of EPN-insect host relationship will undoubtedly facilitate similar studies with mammalian parasitic nematodes, which can lead to the identification and characterization of infection factors that interfere with the human immune system.
The EPN nematodes Heterorhabditis sp. and Steinernema sp. can infect a wide range of insects, and their biology has been intensely studied previously. The two nematode parasites differ in their mode of reproduction with Heterorhabditis being self-fertilized and Steinernema undergoing amphimictic reproduction, although recently S. hermaphroditum was shown to reproduce by self-fertilization of hermaphrodites or through parthenogenesis7,8,9. Another difference between Heterorhabditis and Steinernema nematodes is their symbiotic mutualism with two distinct genera of Gram-negative bacteria, Photorhabdus and Xenorhabdus, respectively, which are both potent pathogens of insects. These bacteria are found in the free-living and non-feeding infective juvenile (IJ) stage of the EPN, which detect susceptible hosts, gain access to the insect hemocoel where they release their associated bacteria that replicate rapidly, and colonize insect tissues. Both the EPN and their bacteria produce virulence factors that disarm insect defenses and impair homeostasis. Following insect death, the nematode IJs develop to become adult EPN and complete their life cycle. A new cohort of IJs formed in response to food deprivation and overcrowding within the insect cadaver finally emerges in the soil to hunt suitable hosts9,10,11,12.
Here, an efficient protocol for maintaining, amplifying and genetically manipulating EPN nematodes is described. In particular, the protocol outlines the replication of symbiotic H. bacteriophora and S. carpocapsae IJs, the generation of axenic nematode IJs, the production of H. bacteriophora hermaphrodites for microinjection, the preparation of the dsRNA, and the microinjection technique. These methods are essential for understanding the molecular basis of nematode pathogenicity and host anti-nematode immunity.

<table><tbody><tr><td>Agarose</td><td>VWR</td><td>97062-244</td><td></td></tr><tr><td>Ambion Megascript T7 Kit</td><td>Thermo Fisher Scientific</td><td>AM1333</td><td></td></tr><tr><td>Ampicillin</td><td>Fisher Scientific</td><td>611770250</td><td></td></tr><tr><td>Cell culture flask T25</td><td>Fisher Scientific</td><td>156367</td><td></td></tr><tr><td>Cell culture flask T75</td><td>Fisher Scientific</td><td>156499</td><td></td></tr><tr><td>ChoiceTaq Mastermix</td><td>Denville Scientific</td><td>C775Y42</td><td></td></tr><tr><td>Corn oil</td><td>VWR</td><td>470200-112</td><td></td></tr><tr><td>Corn syrup</td><td>MP Biomedicals/VWR</td><td>IC10141301</td><td></td></tr><tr><td>Culture tube 10 mL</td><td>Fisher Scientific</td><td>14-959-14</td><td></td></tr><tr><td>Eppendorf Femtotips Microloader Tips</td><td>Eppendorf</td><td>E5242956003</td><td></td></tr><tr><td>Ethanol</td><td>Millipore-Sigma</td><td>E7023</td><td></td></tr><tr><td>Falcon tube 50 mL</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Femtojet Microinjector</td><td>Eppendorf</td><td>5252000021</td><td></td></tr><tr><td>Filter paper</td><td>VWR</td><td>28320-100</td><td></td></tr><tr><td>Galleria mellonella waxorms</td><td>Petco</td><td>-</td><td></td></tr><tr><td>Glass coverslip</td><td>Fisher Scientific</td><td>12-553-464</td><td>50 x 24 mm</td></tr><tr><td>Halocarbon Oil 700</td><td>Sigma</td><td>H8898</td><td></td></tr><tr><td>Inoculating loop</td><td>VWR</td><td>12000-806</td><td></td></tr><tr><td>Kanamycin</td><td>VWR</td><td>97062-956</td><td></td></tr><tr><td>Kwik-Fil Borosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>1B100F-3</td><td>1.0 mm</td></tr><tr><td>LB Agar</td><td>Fisher Scientific</td><td>BP1425-500</td><td>LB agar miller powder 500 g</td></tr><tr><td>LB Broth</td><td>Fisher Scientific</td><td>BP1426-500</td><td>LB broth miller powder 500 g</td></tr><tr><td>Leica DM IRB Inverted Research Microscope</td><td>Microscope Central</td><td>-</td><td></td></tr><tr><td>MacConkey medium</td><td>Millipore-Sigma</td><td>M7408-250G</td><td></td></tr><tr><td>MEGAclear Transcription Clean-Up Kit</td><td>Thermo Fisher Scientific</td><td>AM1908</td><td></td></tr><tr><td>Microcentrifuge tube</td><td>VWR</td><td>76332-064</td><td>1.5 ml</td></tr><tr><td>NanoDrop 2000 Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Needle syringe</td><td>VWR</td><td>BD305155</td><td>22G</td></tr><tr><td>Nutrient broth</td><td>Millipore-Sigma</td><td>70122-100G</td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-076</td><td></td></tr><tr><td>Partitioned Petri dish</td><td>VWR</td><td>490005-212</td><td></td></tr><tr><td>PBS</td><td>VWR</td><td>97062-732</td><td>Buffer PBS tablets biotech grade 200 tab</td></tr><tr><td>PCR primers</td><td>Azenta</td><td>-</td><td></td></tr><tr><td>Pestle</td><td>Millipore-Sigma</td><td>BAF199230001</td><td>Bel-Art Disposable Pestle</td></tr><tr><td>Petri dish 6 cm</td><td>VWR</td><td>25384-092</td><td>60 x 15 mm</td></tr><tr><td>Petri dish 10 mm</td><td>VWR</td><td>10799-192</td><td>35 x 10 mm</td></tr><tr><td>Proteose Peptone #3</td><td>Thermo Fisher Scientific</td><td>211693</td><td></td></tr><tr><td>Yeast extract</td><td>Millipore-Sigma</td><td>Y1625</td><td></td></tr></tbody></table>

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.

To assess the status of H. bacteriophora nematodes that have gone through the axenization, the presence or absence of P. luminescens bacterial colonies in IJs was determined. To do this, a pellet of approximately 500 IJs that had been previously surface sterilized and homogenized in PBS was collected. The positive control treatment consisted of a pellet of approximately 500 IJs from the nematode culture containing symbiotic P. luminescens bacteria. The pellets of axenized and positive control nematodes were homogenized in 1x PBS. The homogenates were pipetted onto agar and spread to facilitate the growth of individual colonies. The agar plates were incubated at 28 &#176;C for 24 h. The next day, the occurrence of P. luminescens colonies (CFU) was observed. As expected, H. bacteriophora from the stock culture carried symbiotic P. luminsecens cells; however, nematodes devoid of their associated P. luminecens bacteria were axenic and stored separately for future experimentation (Figure 6).
Knockdown of nol-5 gene expression was used as an example to show the efficacy of RNAi using the microinjection process. Microinjection was performed in the parental generation and the effect was observed in the F1 progeny. Knockdown of nol-5 using RNAi microinjection resulted in absence of germline in the gonad of 60% of the progeny (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63885/63885fig01.jpg" /> 
Figure 1: Generation of infective juveniles (free living stage of entomopathogenic nematodes). (a) Infection of Galleria mellonella insect larvae (kept in Petri dish) with nematode infective juveniles (kept in tissue culture flask). (b) Folding of a piece of filter paper for preparation of a water trap. (c) The arrangement of a water trap of entomopathogenic nematodes with dead Galleria mellonella insect larvae containing infective juveniles (12 days post nematode infection). The new generation of infective juveniles will leave the dead insects and move to the water, which is then stored into a tissue culture flask. Images are made using BioRender graphic software (https://biorender.com). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63885/63885fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63885/63885fig02.jpg" /> 
Figure 2: Injection of Galleria mellonella insect larvae with the bacterium strain Photorhabdus temperata Ret16. Waxworms are kept in a Petri dish, and they are immobilized through cold treatment (i.e., they are placed on ice for a few minutes). The posterior part of the insect body is pressed with a pair of forceps to create a swollen surface which is suitable for injection. The injection angle is shallow to prevent injuring internal insect tissues, which would lead to insect death due to careless handling. Images are made using BioRender graphic software (https://biorender.com). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63885/63885fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63885/63885fig03.jpg" /> 
Figure 3: Generation of axenic Steinernema carpocapsae entomopathogenic nematodes. One half of a divided Petri dish contains a lawn of Xenorhabdus nematophila &#916;rpoS bacteria on lipid agar and infective juveniles of S. carpocapsae nematodes. This in vitro method induces the infective juveniles to become adult nematodes, which will later produce eggs that will hatch to give rise to the new generation of the parasites. When a large number of newly generated S. carpocapsae infective juveniles appear in this part of the divided Petri dish, water is added to the other half of the dish together with a piece of filter paper to facilitate the migration of the nematodes. Images are made using BioRender graphic software (https://biorender.com) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63885/63885fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63885/63885fig04.jpg" /> 
Figure 4: Breaking the needle tip. The tip of the needle is usually closed. The proper flow of the nucleic acid solution is checked before performing the actual injection. On a glass slide, place a drop of halocarbon oil. Place a capillary tube, used for making the needle, vertically as shown in the figure. Bring the capillary to focus at 40x and gently bring the needle down in plane with the capillary. Gently touch the needle tip to the capillary. This creates an opening for the smooth flow of the dsRNA. If no fluid is coming out, perform a quick on-off nitrogen flow using the foot actuator. The pressure from the nitrogen and the contact of the needle tip with the capillary will break the needle. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63885/63885fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63885/63885fig05.jpg" /> 
Figure 5: Site of injection. The gonad of H. bacteriophora migrates dorsally from the ventral vulval position and then migrates back around to the ventral position. The migrating arms cross each other near the vulva and extend beyond the vulva on either side. At around 48 h, the extending arms of the gonad of a young adult, grown from IJ, are visible near the vulva. Microinjection is performed at this position. Scale bar: 0.1 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63885/63885fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63885/63885fig06.jpg" /> 
Figure 6: Validation of Heterorhabditis bacteriophora axenization. The success of the axenization procedure was estimated by confirming the absence of Photorhabdus luminescens bacterial cells in treated H. bacteriophora infective juveniles. For this, a pellet of approximately 500 surface-sterilized worms from the stock culture (symbiotic) or worms which had undergone the axenization process (axenic) were homogenized. Then, the homogenate was spread onto agar plates and following a 24 h incubation, the appearance of bacterial colonies (colony forming units, CFU) was monitored. Lack of bacterial colonies on the plates suggests that H. bacteriophora nematodes are free of their P. luminescens symbiotic bacteria. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63885/63885fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63885/63885fig07.jpg" /> 
Figure 7: RNAi mediated knockdown of nol-5 gene. RNAi mediated knockdown of H. bacteriophora nol-5 gene results in a phenotype with no germline. The progeny of a wild type, non-injected H. bacteriophora nematode (a) contains eggs in its gonads. The progeny of a wild type, nol-5 RNAi injected H. bacteriophora nematode (b) contains no eggs in its empty gonads due to the knockdown of the nol-5 RNA. Scale bar: 0.1 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63885/63885fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Culturing and Genetically Manipulating Entomopathogenic Nematodes at JoVE.com

Culturing and Genetically Manipulating Entomopathogenic Nematodes

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 ...

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Immunology and Infection

3.2K Views.  The George Washington University. 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.

Video: Culturing and Genetically Manipulating Entomopathogenic Nematodes

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

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

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

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

Biology

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 ...

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 ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

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 ...

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. The increase in pest resistance to chemical insecticides, the growing concerns regarding the negative effects of insecticides on human health, and the environmental pollution from pesticides have led to a global drive to find novel sustainable strategies for crop protection and pest control. Previously, attempts to mass culture such entomopathogenic fungi (EPF) species as Beauveria bassiana have been conducted. However, only limited attempts have been conducted to mass culture Metarhizium robertsii and M. pinghaense for use against insect pests. This study aimed to mass-produce a sufficient number of resilient infective propagules of South African isolates of M. robertsii and M. pinghaense for commercial application. Three agricultural grain products, flaked oats, flaked barley, and rice, were used as the EPF solid fermentation substrates. Two inoculation methods, conidial suspensions and the liquid fungal culture of blastospores were used to inoculate the solid substrates. Inoculation using conidial suspensions was observed to be relatively less effective, as increased levels of contamination were observed on the solid substrates relative to when using the blastospore inoculation method. Flaked oats were found not to be a suitable growth substrate for both M. robertsii and M. pinghaense, as no dry conidia were harvested from the substrate. Flaked barley was found to favor the production of M. robertsii conidia over that of M. pinghaense, and an average of 1.83 g &plusmn; 1.47 g of dry M. robertsii conidia and zero grams of M. pinghaense conidia was harvested from the substrate. Rice grains were found to favor the conidial mass production of both M. pinghaense and M. robertsii isolates, with an average of 8.2 g &plusmn; 4.38 g and 6 g &plusmn; 2 g harvested from the substrate, respectively.

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi have gained importance as the biological control agents of agricultural insect pests. In this study, the mass production of a sufficient number of resilient infective propagules of South African isolates of both Metarhizium robertsii and M. pinghaense for commercial application against insect pests was successfully conducted using agricultural grain products.

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. ...

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 ...

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Summary

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Chapters in this video

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

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

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

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

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

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

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

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

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