RNAi-mediated gene knockdown by microinjection in the model entomopathogenic nematode Heterorhabditis bacteriophora

Abstract

BackgroundParasitic nematodes threaten the health of humans and livestock and cause a major financial and socioeconomic burden to modern society. Given the widespread distribution of diseases caused by parasitic nematodes there is an urgent need to develop tools that will elucidate the genetic complexity of host-parasite interactions. Heterorhabditis bacteriophora is a parasitic nematode that allows simultaneous monitoring of nematode infection processes and host immune function, and offers potential as a tractable model for parasitic nematode infections. However, molecular tools to investigate these processes are required prior to its widespread acceptance as a robust model organism. In this paper we describe microinjection in adult H. bacteriophora as a suitable means of dsRNA delivery to knockdown gene transcripts.MethodsRNA interference was used to knockdown four genes by injecting dsRNA directly into the gonad of adult hermaphrodite nematodes. RNAi phenotypes were scored in the F1 progeny on the fifth day post-injection, and knockdown of gene-specific transcripts was quantified with real-time quantitative RT-PCR (qRT-PCR).ResultsRNAi injection in adult hermaphrodites significantly decreased the level of target transcripts to varying degrees when compared with controls. The genes targeted by RNAi via injection included cct-2, nol-5, dpy-7, and dpy-13. In each case, RNAi knockdown was confirmed phenotypically by examining the progeny of injected animals, and also confirmed at the transcriptional level by real-time qRT-PCR.ConclusionsHere we describe for the first time the successful use of microinjection to knockdown gene transcripts in H. bacteriophora. This technique can be used widely to study the molecular basis of parasitism.

Identification of candidate infection genes from the model entomopathogenic nematode Heterorhabditis bacteriophora

“…Due to its life cycle and recent studies, the possibility of developing and using H. bacteriophora as a model for parasitic infections has grown. Basic advances in the development of a molecular toolbox and the publication of its genome provides a way for manipulating and identifying the function of parasitic genes [5, 8, 13, 19]. In order to create a smaller list of genes involved in infection for future investigation, we performed RNA-seq on H. bacteriophora strain TT01 IJs incubated in hemolymph plasma for 9 h. After differential expression analysis, a total of 1641 genes were identified as being differentially expressed after a 9 h exposure to hemolymph plasma.…”

“…Even though a great deal has been learned about nematode biology and development using the free-living nematode Caenorhabditis elegans , it is not a parasite and thus does not allow for relevant investigations of PN infection mechanisms. For this reason, interest in developing Heterorhabditis bacteriophora into a model organism in order to study nematode parasitism has recently grown [5–8]. …”

“…Additionally, advanced molecular tools (e.g. gene silencing by RNAi) are being developed for H. bacteriophora as well as the recent publication of its genome, making H. bacteriophora potentially an excellent alternative model for nematode parasitism [5, 8, 12–14]. Furthermore, the ability to propagate H. bacteriophora in the immunology model Drosophila melanogaster , allows the study of host responses to PN infection mechanisms [15–17].…”

“…While the basic tools to develop H. bacteriophora as a model organism have been or are in the process of being developed [8, 13], genes directly involved in parasitism are still poorly understood. Studies examining the transcriptome of H. bacteriophora have been performed, but our results described here is the first study, to our knowledge, to utilize advanced next-generation sequencing technologies and the published H. bacteriophora genome to examine the transcriptional program during host invasion [18, 19].…”

Olfactory circuits and behaviors of nematodes

“…These include the ability to generate transgenic nematodes by gonadal microinjection and the ability to conduct genome editing using the CRISPR/Cas9 system [63]. In addition, RNAi has been used successfully with some parasitic nematodes [63,64]. These techniques will enable studies of the neurons and circuits underlying the host-seeking behaviors of parasitic nematodes.…”