The inhibitory effect of EPNP had not been tested before on the development of nematode larvae. In malaria parasites, however, this protease inhibitor disintegrates gametocyte membranes, and it has been used frequently as an inhibitor of peptidases of the A1 family. This is particularly interesting, since the other aspartic protease inhibitor tested, pepstatin A, was neither effective in our model nor in related nematodes, probably due to its limited half-life. Since the development of nematode larvae and the sophisticated moulting process require a series of structural, biochemical, metabolic and physiological changes, we assumed that the proteins essential for fundamental developmental processes in development- inhibited larvae are less abundantly expressed compared with those of uninhibited controls in which normal development occurred. Thus, protein spots that were significantly differentially expressed between development-inhibited and control larvae were subjected to mass spectrometric and bioinformatic analyses. Using this AbK approach, we identified 29 spots representing 22 different proteins. Several spots located at different positions in the same gel were inferred to be distinct protein isoforms or the same protein with varying post-translational modifications. Furthermore, 27 out of 29 identified spots were encoded in O. dentatum contigs derived from ESTs generated by 454 sequencing and analyzed using an advanced bioinformatic pipeline for the interference of key biological processes, such as development and moulting. The O. dentatum contigs were further subjected to homology-based functional annotation. All O. dentatum amino acid sequences characterised had homologous proteins in other nematodes, including species from the orders Strongylida, Rhabditida, Spirurida, and Ascaridida. The proteins identified and annotated included structural and cytoskeletal proteins, enzymes involved in metabolism, stress-associated peptides, regulatory and interacting peptides and one protease-inhibiting protein. The expression of the vast majority of the 22 proteins identified was down-regulated in the development-inhibited group compared with controls, whereas three proteins were shown to be up-regulated. We functionally annotated 19 of 22 protein sequences using GO and established pathway associations for 14 of 22 sequences in KEGG. The annotation of the 22 proteins revealed specific roles in larval developmental processes for intermediate filament protein B, HSP-70, troponin T, HSP-60, calreticulin, actin, fructose-bisphosphate aldolase, tropomyosin, malate dehydrogenase, RACK-1, A 61603 hydrobromide pyruvate dehydrogenase E1 and 14-3-3 protein. The expression of all 12 proteins was down-regulated in the development-inhibited larvae compared with controls.