Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.
- MeSH
- Caenorhabditis elegans genetika MeSH
- cholesteny farmakologie MeSH
- delece genu MeSH
- druhová specificita MeSH
- genová ontologie MeSH
- Haemonchus účinky léků genetika růst a vývoj MeSH
- larva MeSH
- messenger RNA genetika metabolismus MeSH
- mikro RNA biosyntéza genetika MeSH
- proteiny Caenorhabditis elegans genetika MeSH
- receptor inzulinu genetika MeSH
- RNA helmintů biosyntéza genetika MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UDP-glycosyltransferases (UGT), catalysing conjugation of UDP-activated sugar donors to small lipophilic chemicals, are widespread in living organisms from bacteria to fungi, plant, or animals. The progress of genome sequencing has enabled an assessment of the UGT multigene family in Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite of small ruminants. Here we report 32 putative UGT genes divided into 15 UGT families. Phylogenetic analysis in comparison with UGTs from Caenorhabditis elegans, a free-living model nematode, revealed several single member homologues, a lack of the dramatic gene expansion seen in C. elegans, but also several families (UGT365, UGT366, UGT368) expanded in H. contortus only. The assessment of constitutive UGT mRNA expression in H. contortus adults identified significant differences between females and males. In addition, we compared the expression of selected UGTs in the drug-sensitive ISE strain to two benzimidazole-resistant strains, IRE and WR, with different genetic backgrounds. Constitutive expression of UGT368B2 was significantly higher in both resistant strains than in the sensitive strain. As resistant strains were able to deactivate benzimidazole anthelmintics via glycosylation more effectively then the sensitive strain, UGT368B2 enhanced constitutive expression might contribute to drug resistance in H. contortus.
- MeSH
- anthelmintika farmakologie MeSH
- benzimidazoly farmakologie MeSH
- Caenorhabditis elegans enzymologie genetika MeSH
- exprese genu MeSH
- fylogeneze * MeSH
- glykosylace MeSH
- glykosyltransferasy chemie klasifikace genetika MeSH
- Haemonchus účinky léků enzymologie genetika MeSH
- léková rezistence genetika MeSH
- mapování chromozomů MeSH
- multigenová rodina MeSH
- nemoci ovcí parazitologie MeSH
- ovce MeSH
- sexuální faktory MeSH
- uridindifosfát genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Sheep farmers in the UK rely on strategic anthelmintic use to treat and control gastrointestinal roundworms in their flocks. However, resistance to these drugs is now widespread and threatens the sustainability of sheep production. The mechanisms underlying resistance to the most commonly used class, the macrocyclic lactones, are not known and sensitive diagnostic tools based on molecular markers are not currently available. This prohibits accurate surveillance of resistance or assessment of strategies aimed at controlling its spread. In this study, we examined four UK field populations of Haemonchus contortus, differing in macrocyclic lactone treatment history, for evidence of selection at 'candidate gene' loci identified as determining macrocyclic lactone resistance in previously published research. Individual worms were genotyped at Hc-lgc-37, Hc-glc-5, Hc-avr-14 and Hc-dyf-7, and four microsatellite loci. High levels of polymorphism were identified at the first three candidate gene loci with remarkably little polymorphism at Hc-dyf-7. While some between-population comparisons of individual farms with and without long-term macrocyclic lactone use identified statistically significant differences in allele frequency and/or fixation index at the Hc-lgc-37, Hc-glc-5 or Hc-avr-14 loci, we found no consistent evidence of selection in other equivalent comparisons. While it is possible that different mechanisms are important in different populations or that resistance may be conferred by small changes at multiple loci, our findings suggest that these are unlikely to be major loci conferring macrocyclic lactone resistance on UK farms or suitable for diagnostic marker development. More powerful approaches, using genome-wide or whole genome sequencing, may be required to define macrocyclic lactone resistance loci in such genetically variable populations.
- MeSH
- frekvence genu MeSH
- genetická variace MeSH
- genotypizační techniky MeSH
- Haemonchus účinky léků genetika MeSH
- hemonchóza farmakoterapie parazitologie veterinární MeSH
- laktony farmakologie terapeutické užití MeSH
- léková rezistence genetika MeSH
- makrocyklické laktamy farmakologie terapeutické užití MeSH
- nemoci ovcí farmakoterapie parazitologie MeSH
- ovce MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- vazebná nerovnováha MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Spojené království MeSH
The aim of this work was to identify reliable reference genes for expression studies in adult Haemonchus contortus. Eleven candidate genes were identified and the stability of their expression was assessed in adult males and females of two genetically divergent H. contortus isolates: drug-susceptible (ISE) and multi-drug-resistant (WR). Five genes with the most stable expression patterns were further assessed for suitability as reference genes in anthelmintic-treated H. contortus adults versus non-treated controls. We identified important differences in the expression of a number of candidate genes in anthelmintic-treated samples, confirming the need for careful validation of control genes for such experiments. We propose the use of multiple reference genes for expression studies in this species and found gpd, ama and far most suitable for adult H. contortus.
- MeSH
- anthelmintika farmakologie MeSH
- Haemonchus účinky léků genetika MeSH
- kvantitativní polymerázová řetězová reakce metody normy MeSH
- referenční standardy * MeSH
- stanovení celkové genové exprese metody normy MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH