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DAF-7 is the ligand of the TGF-β pathway that, in conjunction with the insulin-like and guanylyl cyclase pathways, controls entry into dauer development in Caenorhabditis elegans. Proposed orthologues of Ce-daf-7 have been identified in several species of parasitic nematodes and demonstrate an expression pattern that is consistent between parasitic nematode genera but different to that of Ce-daf-7. This variation in expression pattern is consistent with the current paradigm in evolutionary developmental biology: that regulatory rather than functional change is the primary source of phenotypic diversity. In this work we investigated the proposed orthology of a daf-7 like sequence obtained from Parastrongyloides trichosuri, Pt-daf-7, to Ce-daf-7 via transformation rescue of a C. elegans daf-7 mutant with Pt-daf-7 coding regions. We also investigated further the difference in expression pattern of Pt-daf-7 both by fusing a Pt-daf-7 promoter to a Ce-daf-7 coding region and to a gfp reporter gene. We found that Pt-daf-7 was unable to complement a C. elegans daf-7 mutant, even when reduced to the smallest functional TGF-β unit possible, the ligand domain, and that this failure appears to be the result of gene silencing. Furthermore, we show that although the Pt-daf-7 promoter is active later in development than the Ce-daf-7 promoter and most likely active in the correct neurons, a Ce-daf-7 coding region under control of a Pt-daf-7 promoter failed to rescue. Together, these results suggest that, if the free-living nematode developmental pathways, such as the DAF-7 TGF- β pathway, have been co-opted during the evolution of parasitism, this co-option has been both at the protein level and in the control of their transcription.

Germline transformation of a parasitic nematode of mammals has proven to be an elusive goal. We report here the heritable germline transformation of Parastrongyloides trichosuri, a nematode parasite whose natural hosts are Australian possums of the genus Trichosurus. This parasite can undergo multiple free-living life cycles and these replicative cycles can be maintained indefinitely in the laboratory. Transformation was achieved by microinjection of DNA into the ovary syncytium of either free-living or parasitic adult females. By selecting for the transgenic progeny of successive free-living life cycles, it was possible to establish and maintain transgenic lines. All three transgenic lines tested were shown capable of establishing patent infections in possums and to transmit the functional transgene to their progeny. The transgene, driven by the Pt hsp-1 promoter, was constitutively expressed in intestinal cells at all stages of both parasitic and free-living life cycles, although gene silencing appears to occur in some transgenic progeny. This is the first report of heritable transgenesis in a parasitic nematode of a mammal and we discuss a variety of previously inaccessible experimental avenues that will now be possible with this powerful model system.