Name: culturing and genetically manipulating entomopathogenic nematodes
Uploaded: 2022-03-31T15:00:00
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.

1. Production of symbiotic nematode infective juveniles
<ol>
	<li>Cover a Petri dish (10 cm) with a piece of filter paper and add approximately 10-15 Galleria mellonella larvae (Figure 1A).</li>
	<li>Using a pipette, dispense 2 mL of water containing about 25-50 IJs per 10 &#181;L suspension onto the waxworms. Store the Petri dish in a cabinet at room temperature.</li>
	<li>Depending on the moisture of the filter paper, add 1-2 mL of water every 2 days. Waxworms i...

<ol>
	<li>Lacey, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+pathogens+as+biological+control+agents:+Back+to+the+future.">Insect pathogens as biological control agents: Back to the future.</a> Journal of Invertebrate Pathology. 132, 1-41 (2015).</li><li>Ozakman, Y., Eleftherianos, 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+infection+and+antinematode+immunity+in+Drosophila.">Nematode infection and antinematode immunity in Drosophila.</a> Trends in Parasitology. 37 (11), 1002-1013 (2021).</li><li>Kenney, E., Hawdon, J. M., O'Halloran, D. M., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+virulence+factors+from+Heterorhabditis+bacteriophora+highlight+its+utility+as+a+model+parasite+among+Clade+V+nematodes.">Secreted virulence factors from Heterorhabditis bacteriophora highlight its utility as a model parasite among Clade V nematodes.</a> International Journal of Parasitology. 51 (5), 321-325 (2021).</li><li>Bobardt, S. D., Dillman, A. R., Nair, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+faces+of+nematode+infection:+Virulence+and+immunomodulatory+molecules+from+nematode+parasites+of+mammals,+insects+and+plants.">The two faces of nematode infection: Virulence and immunomodulatory molecules from nematode parasites of mammals, insects and plants.</a> Frontiers in Microbiology. 11, 2983 (2020).</li><li>Castillo, J. C., Reynolds, S. E., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+immune+responses+to+nematode+parasites.">Insect immune responses to nematode parasites.</a> Trends in Parasitology. 27 (12), 537-547 (2011).</li><li>Eleftherianos, I., Heryanto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+insights+into+the+insect+immune+response+to+nematode+infection.">Transcriptomic insights into the insect immune response to nematode infection.</a> Genes. 12 (2), 202 (2021).</li><li>Ciche, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+and+genome+of+Heterorhabditis+bacteriophora.">The biology and genome of Heterorhabditis bacteriophora.</a> WormBook. , 1-9 (2007).</li><li>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=Partners+in+crime:+symbiont-assisted+resource+acquisition+in+Steinernema+entomopathogenic+nematodes.">Partners in crime: symbiont-assisted resource acquisition in Steinernema entomopathogenic nematodes.</a> Current Opinion in Insect Science. 32, 22-27 (2019).</li><li>Cao, M., Schwartz, H. T., Tan, C. -. H., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+entomopathogenic+nematode+Steinernema+hermaphroditum+is+a+self-fertilizing+hermaphrodite+and+a+genetically+tractable+system+for+the+study+of+parasitic+and+mutualistic+symbiosis.">The entomopathogenic nematode Steinernema hermaphroditum is a self-fertilizing hermaphrodite and a genetically tractable system for the study of parasitic and mutualistic symbiosis.</a> Genetics. 220 (1), (2021).</li><li>Goodrich-Blair, H., Clarke, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutualism+and+pathogenesis+in+Xenorhabdus+and+Photorhabdus:+two+roads+to+the+same+destination.">Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination.</a> Molecular Microbiology. 64 (2), 260-268 (2007).</li><li>Abd-Elgawad, M. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photorhabdus+spp.:+An+overview+of+the+beneficial+aspects+of+mutualistic+bacteria+of+insecticidal+nematodes.">Photorhabdus spp.: An overview of the beneficial aspects of mutualistic bacteria of insecticidal nematodes.</a> Plants. 10 (8), 1660 (2021).</li><li>Dreyer, J., Malan, A. P., Dicks, L. M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+of+the+genus+Xenorhabdus,+a+novel+source+of+bioactive+compounds.">Bacteria of the genus Xenorhabdus, a novel source of bioactive compounds.</a> Frontiers in Microbiology. 9, 3177 (2018).</li><li>Hallem, E. A., Rengarajan, M., Ciche, T. A., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nematodes,+bacteria,+and+flies:+a+tripartite+model+for+nematode+parasitism.">Nematodes, bacteria, and flies: a tripartite model for nematode parasitism.</a> Current Biology. 17 (10), 898-904 (2007).</li><li>Castillo, J. C., Shokal, U., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+infecting+Drosophila+adult+flies+with+insect+pathogenic+nematodes.">A novel method for infecting Drosophila adult flies with insect pathogenic nematodes.</a> Virulence. 3 (3), 339-347 (2012).</li><li>Castillo, J. C., Shokal, U., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+gene+transcription+in+Drosophila+adult+flies+infected+by+entomopathogenic+nematodes+and+their+mutualistic+bacteria.">Immune gene transcription in Drosophila adult flies infected by entomopathogenic nematodes and their mutualistic bacteria.</a> Journal of Insect Physiology. 59 (2), 179-185 (2013).</li><li>Eleftherianos, I., Joyce, S., Ffrench-Constant, R. H., Clarke, D. J., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+tri-trophic+interaction+between+insects,+nematodes+and+Photorhabdus.">Probing the tri-trophic interaction between insects, nematodes and Photorhabdus.</a> Parasitology. 137 (11), 1695-1706 (2010).</li><li>Nielsen-LeRoux, C., Gaudriault, S., Ramarao, N., Lerelcus, D., Givaudan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+insect+pathogen+bacteria+Bacillus+thuringiensis+and+Xenorhabdus/Photorhabdus+occupy+their+hosts.">How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts.</a> Current Opinion in Microbiology. 15 (3), 220-231 (2012).</li><li>Waterfield, N. R., Ciche, T., Clarke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photorhabdus+and+a+host+of+hosts.">Photorhabdus and a host of hosts.</a> Annual Review of Microbiology. 63, 557-574 (2009).</li><li>Ozakman, Y., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+interactions+between+Drosophila+and+the+pathogen+Xenorhabdus.">Immune interactions between Drosophila and the pathogen Xenorhabdus.</a> Microbiological Research. 240, 126568 (2020).</li><li>Yadav, S., Shokal, U., Forst, S., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+generating+axenic+entomopathogenic+nematodes.">An improved method for generating axenic entomopathogenic nematodes.</a> BMC Research Notes. 8 (1), 1-6 (2015).</li><li>Mitani, D. 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>

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 b...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 n...

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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