Identification of mRNAs that are differentially expressed between serum-stimulated and non-stimulated A. caninum L3

Third-stage larvae (L3s) of the canine hookworm, Ancylostoma caninum, endure a period of arrested development preceding transmission to a host. Many of the mRNAs up-regulated at this stage are likely to encode proteins that act at the host-parasite interface and facilitate the transition from a free-living to a parasitic larva. The initial phase of the infection of a mammalian host by A. caninum L3s (herein termed “activation”) can be mimicked in vitro by culturing L3s in serum-containing medium. The mRNAs differentially transcribed between activated and non-activated L3s were identified by suppression subtractive hybridisation (SSH). The analysis of these mRNAs on a custom oligonucleotide microarray printed with the SSH ESTs and publicly available A. caninum ESTs (non-subtracted) yielded a total of 602 differentially expressed mRNAs, of which the most highly represented sequences (27) encoded products belonging to the pathogenesis-related protein (PRP) superfamily and different mechanistic classes of proteases. Comparison of these A. caninum mRNAs with those of Caenorhabditis elegans larvae exiting from developmental (dauer) arrest demonstrated unexpectedly large differences with respect to gene ontology profiles. C. elegans L3 exiting developmental arrest up-regulated the expression of collagens and other (mostly intracellular) molecules involved in growth and development. Such mRNAs are virtually absent from activated hookworm larvae, and instead are represented by an inordinately large number of mRNAs encoding extracellular proteins, suggesting that many of the activation-associated hookworm mRNAs are involved in host-parasite interactions. The near absence of mRNAs associated with reproduction, growth and development among activated hookworm L3s probably reflects their ability to further arrest (i.e. undergo hypobiosis) in tissues of non-permissive hosts or in the external environment when conditions for transmission are unfavourable. Although this should not necessarily invalidate C. elegans dauer exit as a model for hookworm activation, it provides substantial information on the limitations of this free-living nematode as a model organism for the transition of nematode larvae from a free-living to a parasitic state. Keywords: Comparative transcriptomic hybridisation RNA was extracted from two different groups of Ancylostoma caninum L3 that were separated in time and geography (Populations III and IV). From population III, two separate in vitro activation assays were performed. This yielded two sets of RNA from activated worms and two sets of RNA from non-activated worms. Population IV yielded one sample of RNA from activated worms and one sample from non-activated L3. In total, there were three samples of RNA from activated L3 and three from non-activated L3 representing two biological replicates and one technical replicate for activation. For each hybridization, labelled RNA from activated and non-activated L3 (within a biological and technical replicate) were compared. This comparison was also made using the same samples with reciprocal labeling (dye-swap). Lastly, two self-hybridisations were performed. The first compared RNA from Activated A. caninum L3 (population III) that were labelled separately with Cy3 and Cy5. The same self hybridisation was performed for Non-activated A. caninum L3 (population IV). Non-activated A. caninum L3 served as the reference sample for all non-self hybridisations.