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.
Highlights
Parasitic nematodes threaten the health of humans and livestock and cause a major financial and socioeconomic burden to modern society
In the present study we describe RNA interference (RNAi) in H. bacteriophora by injecting double-stranded RNA (dsRNA) into the gonads of the adult hermaphrodite
We tested the efficacy of dsRNA delivery by microinjection on four H. bacteriophora genes with known RNAi phenotypes
Summary
Parasitic nematodes threaten the health of humans and livestock and cause a major financial and socioeconomic burden to modern society. Diseases caused by parasitic nematodes are a major concern, resulting in human health and socioeconomic consequences [1, 2]. An important interaction between the parasite and its potential host is the active avoidance of the host immune system by the parasite [11] Understanding this interaction at the molecular level provides useful insight into pathogen virulence and host immunity [12, 13]. Advances in understanding these mechanisms require powerful molecular tools to investigate gene function, which are unavailable for most parasitic nematodes. Animal parasitic nematodes in which RNAi has been tested include Nippostrongylus brasiliensis [20, 21], Brugia malayi [22, 23], Onchocerca volvulus [24], Litomosoides sigmodontis [25], Ascaris suum [26, 27], Trichostrongylus colubriformis [28], Haemonchus contortus [29,30,31], Ostertagia ostertagi [32], Teladorsagia circumcincta [33], Trichinella spiralis [34], Heligmosomoides polygyrus [35] and Heterorhabditis bacteriophora [36, 37]
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