Culturing and Genetically Manipulating Entomopathogenic Nematodes

Summary

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.

Abstract

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.

Introduction

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 research^1,2. 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

Protocol

1. Production of symbiotic nematode infective juveniles

1. Cover a Petri dish (10 cm) with a piece of filter paper and add approximately 10-15 Galleria mellonella larvae (Figure 1A).
2. Using a pipette, dispense 2 mL of water containing about 25-50 IJs per 10 µL suspension onto the waxworms. Store the Petri dish in a cabinet at room temperature.
3. Depending on the moisture of the filter paper, add 1-2 mL of water every 2 days. Waxworms i.......

Discussion

Understanding the molecular basis of entomopathogenic nematode infection and insect anti-nematode immunity requires the separation of the parasites from the mutualistically associated bacteria^13,15,16. The entomopathogenic nematodes H. bacteriophora and S. carpocapsae live together with the Gram-negative bacteria P. luminescens and X. nematophila, respectively¹⁷. Both b.......

Materials

Name	Company	Catalog Number	Comments
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