To elucidate the role of transforming growth factor beta (TGF-beta) signalling in the arrest/reactivation pathway of the Ancylostoma caninum hookworm, two parasite-encoded TGF-beta-like ligands were cloned and characterised.
Ac-dbl-1 showed 60% amino acid identity to the Caenorhabditis elegansdbl-1 gene, which regulates growth while
Ac-daf-7 showed 46% amino acid identity to
Ce-daf-7 which regulates arrested development. Exon/intron organisation of the genes for
Ac-dbl-1 and
Ac-daf-7 were different from that of the corresponding C. elegans genes with nine and 10 exons, respectively, and introns ranging in size from 56 to 2556bp. Based on real-time reverse transcriptase (RT)-PCR,
Ac-dbl-1 and
Ac-daf-7 were expressed in all stages tested, i.e. egg, first/second stage larvae (L1/L2), infective third stage larvae (iL3), serum-stimulated third stage larvae (ssL3), and male and female adult worms. Expression of
Ac-dbl-1 peaked in the adult male stage suggesting a similar role to
Ce-dbl-1 in regulating male tail patterning.
Ac-daf-7 expression was at a maximum in the arrested iL3 and reactivated ssL3 stages, which differs from that of
Ce-daf-7 expression and may be unique to parasitic nematodes that have an obligate requirement to undergo developmental arrest. In support of the PCR results, antibodies to the A. caninum TGF-beta-like ligands detected proteins in iL3, ssL3, and adult worm extracts. Immunofluorescent studies showed that
Ac-daf-7 is expressed in the anterior region of the iL3 similar to
Ce-daf-7, which is localised to the ASI chemosensory neurons.