Attempts to establish RNA interference in the parasitic nematode Heligmosomoides polygyrus.
To analyze the potential of RNA interference (RNAi) in the intestinal nematode Heligmosomoides polygyrus, we delivered double-stranded RNA (dsRNA) of tropomyosin to various life stages of the parasite. Three different methods were examined for their potential use. First, feeding of recombinant bacteria that expressed dsRNA did neither result in phenotypical changes of H. polygyrus nor in a significant reduction of tropomyosin mRNA levels. In contrast, feeding of such bacteria to Caenorhabditis elegans elicited the expected phenotypes. Quantification of bacteria ingested by C. elegans and H. polygyrus larvae (L1) revealed that the parasitic worms took up only a fraction of the bacteria ingested by C. elegans. Second, electroporation of L1 failed to transport siRNA through the cuticle and was lethal to the larvae. However, the cuticle of adult worms was penetrated by dye-labeled RNA, but no systemic spreading was observed. Third, soaking of adult H. polygyrus in tropomyosin dsRNA led to a higher proportion of worms showing symptoms of ageing, such as a disintegrated gut and ovaries, but did not induce reduction of tropomyosin mRNA levels. Database analysis revealed that orthologous proteins involved in dsRNA-uptake and -systemic spread in C. elegans are missing in the parasitic nematodes Brugia malayi and Haemonchus contortus, whereas proteins responsible for dsRNA-processing, -amplification and mRNA-regulation are present. Thus, our data indicate that the study of gene function by RNAi in H. polygyrus is limited, possibly due to deficiencies of genes involved in RNA-uptake and spread.
Lendner
Matthias
M
Chair of Molecular Parasitology, Humboldt University Berlin, 10115 Berlin, Germany.
Doligalska
Maria
M
Lucius
Richard
R
Hartmann
Susanne
S
D000375:Aging; D000818:Animals; D017178:Brugia malayi / Q000235:genetics; D017173:Caenorhabditis elegans / Q000235:genetics / Q000382:microbiology; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018274:Electroporation; D005260:Female; D041981:Gastrointestinal Tract / Q000473:pathology; D020869:Gene Expression Profiling; D006189:Haemonchus / Q000235:genetics; D007814:Larva / Q000235:genetics / Q000382:microbiology; D008969:Molecular Sequence Data; D009351:Nematospiroides dubius / Q000235:genetics* / Q000382:microbiology; D010053:Ovary / Q000473:pathology; D034622:RNA Interference; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D014335:Tropomyosin / Q000096:biosynthesis / Q000235:genetics*
D014335:Tropomyosin; D004247:DNA