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 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.
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.
1. Production of symbiotic nematode infective juveniles
2. Production of axenic nematode infective juveniles
NOTE: Axenic nematodes are used because after the nematode-bacteria complex dissociates inside the insect, each mutualistic partner elicits a distinct host immune response5. The mutant strain Ret16 of Photorhabdus temperata is used because these bacteria support the growth of H. bacteriophora but fail to colonize the nematode gut13,14.
3. Raising H. bacteriophora hermaphrodites for microinjection
4. Preparation of dsRNA
5. Microinjection
NOTE: An injection pad is a glass coverslip with a layer of 2% (w/v) agarose on the center. When the worms to be injected are transferred to these pads, the agarose layer will immobilize them for the procedure. Normally, extra pads are kept near the microscope for general use.
6. Microinjection
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 °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).
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). Please click here to view a larger version of this figure.
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). Please click here to view a larger version of this figure.
Figure 3: Generation of axenic Steinernema carpocapsae entomopathogenic nematodes. One half of a divided Petri dish contains a lawn of Xenorhabdus nematophila ΔrpoS 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) Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 ΔrpoS 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/µL to 10 µg/µL were tested. It was found that a concentration of 6 µg/µL 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.
The authors have nothing to disclose.
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'H. laboratories have been supported by George Washington University and Columbian College of Arts and Sciences facilitating funds and Cross-Disciplinary Research Funds.
Agarose | VWR | 97062-244 | |
Ambion Megascript T7 Kit | Thermo Fisher Scientific | AM1333 | |
Ampicillin | Fisher Scientific | 611770250 | |
Cell culture flask T25 | Fisher Scientific | 156367 | |
Cell culture flask T75 | Fisher Scientific | 156499 | |
ChoiceTaq Mastermix | Denville Scientific | C775Y42 | |
Corn oil | VWR | 470200-112 | |
Corn syrup | MP Biomedicals/VWR | IC10141301 | |
Culture tube 10 mL | Fisher Scientific | 14-959-14 | |
Eppendorf Femtotips Microloader Tips | Eppendorf | E5242956003 | |
Ethanol | Millipore-Sigma | E7023 | |
Falcon tube 50 mL | Fisher Scientific | 14-432-22 | |
Femtojet Microinjector | Eppendorf | 5252000021 | |
Filter paper | VWR | 28320-100 | |
Galleria mellonella waxorms | Petco | – | |
Glass coverslip | Fisher Scientific | 12-553-464 | 50 x 24 mm |
Halocarbon Oil 700 | Sigma | H8898 | |
Inoculating loop | VWR | 12000-806 | |
Kanamycin | VWR | 97062-956 | |
Kwik-Fil Borosilicate Glass Capillaries | World Precision Instruments | 1B100F-3 | 1.0 mm |
LB Agar | Fisher Scientific | BP1425-500 | LB agar miller powder 500 g |
LB Broth | Fisher Scientific | BP1426-500 | LB broth miller powder 500 g |
Leica DM IRB Inverted Research Microscope | Microscope Central | – | |
MacConkey medium | Millipore-Sigma | M7408-250G | |
MEGAclear Transcription Clean-Up Kit | Thermo Fisher Scientific | AM1908 | |
Microcentrifuge tube | VWR | 76332-064 | 1.5 ml |
NanoDrop 2000 Spectrophotometer | Thermo Fisher Scientific | ND-2000 | |
Needle syringe | VWR | BD305155 | 22G |
Nutrient broth | Millipore-Sigma | 70122-100G | |
Parafilm | VWR | 52858-076 | |
Partitioned Petri dish | VWR | 490005-212 | |
PBS | VWR | 97062-732 | Buffer PBS tablets biotech grade 200 tab |
PCR primers | Azenta | – | |
Pestle | Millipore-Sigma | BAF199230001 | Bel-Art Disposable Pestle |
Petri dish 6 cm | VWR | 25384-092 | 60 x 15 mm |
Petri dish 10 mm | VWR | 10799-192 | 35 x 10 mm |
Proteose Peptone #3 | Thermo Fisher Scientific | 211693 | |
Yeast extract | Millipore-Sigma | Y1625 |