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Background: Enoyl-CoA hydratase (MAOC) is required for the biosynthesis of the fatty acid-derive side chains of the ascaroside via peroxisome β-oxidation in the free-living nematode Caenorhabditis elegans. The derivative of dideoxy-sugar, ascarylose is used as dauer pheromones or daumones to induce development of the stress-resistant dauer larvae stage., Methods: Hc-maoc-1 gene was obtained by searching the Wellcome Trusts Sanger Institute's H. contortus genomic database. qRT-PCR was performed to analyse the transcriptional levels of Hc-maoc-1 with different developmental stages as templates. IFA was carried out to determine the expression pattern in L3 larvae and micro-injection was used to verify the promoter activity of 5'-flanking region of Hc-maoc-1. Overexpression and RNAi experiments were applied in N 2 strain to ascertain the gene function of Hc-maoc-1., Results: The full-length cDNA of Hc-maoc-1 was 900 bp in length, which contained eight exons separated by seven introns and possessed the Hotdog domain and the MaoC-like domain, together with several other residues and a hydratase 2 motif. It was transcribed throughout the lifecycle and peaked in the fourth-stage larvae (L4) of H. contortus; however, its transcription level decreased in diapausing L4. The protein expression and location of Hc-MAOC-1 were mainly in the intestine of L3 larvae. Overexpression of Ce-maoc-1 and Hc-maoc-1 in C. elegans showed extended lifespan and increased body size. The protein Ce-MAOC-1 and Hc-MAOC-1 were localized in the intestine with a punctate pattern. In C. elegans, knockdown of Ce-maoc-1 conferred shortened lifespan and body lengths, decreased brood size and increased lipid storage., Conclusion: Caenorhabditis elegans was used as a model organism to ascertain the function of Hc-maoc-1 in H. contortus. Our results showed the similar characteristics and functions with Ce-maoc-1 and provided evidences of the potential functions of Hc-maoc-1 in biosynthesis of daumones in H. contortus.

Background: The strongylid nematode Haemonchus contortus is a parasite of major concern for modern livestock husbandry because hostile environmental conditions may induce diapause in the early fourth-stage larvae., Methods: A new gene Hc-daf-22 was identified which is the homologue of Ce-daf-22 and human SCPx. Genome walking and RACE were performed to obtain the whole cDNA and genomic sequence of this gene. Using qRT-PCR with all developmental stages as templates to explore the transcription level and micro-injection was applied to confirm the promoter activity of the 5'-flanking region. Overexpression, rescue and RNA interference experiments were performed in N2, daf-22 mutant (ok 693) strains of C. elegans to study the gene function of Hc-daf-22., Results: The full length gene of Hc-daf-22 (6,939 bp) contained 16 exons separated by 15 introns, and encoded a cDNA of 1,602 bp (533 amino acids, estimated at about 59.3 kDa) with a peak in L3 and L4 in transcriptional level. The Hc-DAF-22 protein was consisted of a 3-oxoacyl-CoA thiolase domain and a SCP2 domain and evolutionarily conserved. The 1,548 bp fragment upstream of the 5'-flanking region was confirmed to have promoter activity compared with 5'-flanking region of Ce-daf-22. The rescue experiment by micro-injection of daf-22 (ok693) mutant strain showed significant increase in body size and brood size in the rescued worms with significantly reduced or completely absent fat granules confirmed by Oil red O staining, indicating that Hc-daf-22 could partially rescue the function of Ce-daf-22. Furthermore, RNAi with Hc-daf-22 could partially silence the endogenous Ce-daf-22 in N2 worms and mimic the phenotype of daf-22 (ok693) mutants., Conclusion: The gene Hc-daf-22 was isolated and its function identified using C. elegans as a model organism. Our results indicate that Hc-daf-22 shared similar characteristics and function with Ce-daf-22 and may play an important role in peroxisomal β-oxidation and the development in H. contortus.

Diapause induced in the early fourth stage of Haemonchus contortus is a strategy to adapt this nematode to hostile environmental conditions. In this study, we identified a new gene, Hc-fau, a homologue of human fau and Caenorhabditis elegans Ce-rps30. Hc-fau encodes two proteins through alternative RNA splicing, Hc-FAUA and Hc-FAUB, consisting of 130 and 107 amino acids, respectively. Hc-FAU possesses a diverged ubiquitin-like (UBiL) protein domain and a conserved ribosome protein S30 domain. The protein is ubiquitously expressed, except in the gonad. However Hc-fau transcripts decrease significantly in diapausing L4s of H. contortus. In C. elegans, knockdown of Ce-rps30 confers an extended lifespan, increased lipid storage in the intestine and shortened body length. These morphological characteristics are comparable with dauer larvae of C. elegans, in which the gonad is condensed considerably. In contrast, a shortened lifespan is observed in C. elegans over-expressing Hc-faua, and especially Hc-faub, with hatching failure detected. The genes of insulin/IGF-1 signalling (IIS), TGF-β, cGMP, dafachronic acid (DA), apoptosis (AP) and fatty acids (FA) metabolism are all down-regulated in Ce-rps30RNAi (RNA interference) worms, except for akt-1 and daf-16. However, daf-16 up-regulation is inconsistent with its target gene down-regulation and the result from a heat stress assay in these worms. Daf-16 RNAi conducted in Ce-rps30 (tm6034/nt1) mutants failed to rescue the worms. The S30 domain stays in the nucleus, while UBiL accumulates in the cytoplasm. Compared with Hc-FAUA, results of UBiL domain and S30 domain over-expression indicate synergism between UBiL and S30 in regulating lifespan and reproduction. These results suggest the potential functions of Hc-fau in regulating larval diapause in H.contortus., (Copyright © 2014. Published by Elsevier Ltd.)

Haemonchus contortus infections in small ruminants are of major economic importance worldwide. Heat shock proteins (HSPs) are a family of molecular chaperones that play important roles in the process of invasion and survival of nematodes. Although HSP70 has been identified in several parasitic nematodes, little is known of its distribution and function in Haemonchus contortus. The aims of this study were to characterize HSP70 from Haemonchus contortus (designed as Hc-hsp70), express Hc-hsp70 and analyse the promoter activity in Caenorhabditis elegans. Bioinformatic analysis revealed that the open reading frame of the Hc-hsp70 cDNA encodes a 646-amino acid peptide, which is highly conserved in comparison to HSP70 in other nematodes. Phylogenetic analysis indicated that H. contortus is closely related to Caenorhabditis. The 5'-flanking region promoted green fluorescence protein (GFP) expression in the intestine in all larval stages and adult with 2 expression patterns in C. elegans. Expression of Hc-hsp70 mRNA transcripts in C. elegans increased following 2, 4, 6 h of heat shock and peaked at 4 h. However, its expression induced down-regulation of hsp-1 of C. elegans. These results suggest that the H. contortus hsp70 might have a similar function to that of C. elegans hsp-1.