In vitro proteolysis of nematode FMRFamide-like peptides (FLPs) by preparations from a free-living nematode (Panagrellus redivivus) and two plant-parasitic nematodes (Heterodera glycines and Meloidogyne incognita).

Proteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyne incognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.

The role of Steinernema feltiae body-surface lipids in host-parasite immunological interactions.

Interactions between entomopathogenic nematodes (Steinernema feltiae) and insect host (Galleria mellonella) immune system were investigated. We focused on the immunosuppressive properties of the parasite cuticle and on its interaction with hemolymph humoral components. Effects of parasite cuticle against host proPO system enzymatic cascade were evaluated a short time after infection. The presence of parasite cuticles decreased both normal and LPS-elicited proPO system activity, suggesting that S. feltiae body surface plays a key role in the early parasitation phase, probably interfering with host proPO activation pathways. The data obtained showed that cuticle lipidic compounds are able to interact with host humoral components, removing them from the hemolymph. The depletion of these molecules, arbitrarily named host-interacting proteins (HIPs), seems to be responsible of the drastic decrease in proPO system activity. Moreover, hemolymph HIPs showed LPS-binding properties and parasite cuticle cross-reacted with anti-LPS antibodies. Finally, we also assessed the involvement of parasite body surface on immunoevasion strategies of S. feltiae against host cell-mediated encapsulation processes. We conclude that S. feltiae body surface is responsible for short-term immunosuppression and immunoevasion processes; since it is able to sequester host hemolymph compounds involved in proPO system activation and this process could be responsible for a molecular disguise strategy against cellular encapsulation.

The FMRFamide-like neuropeptide AF2 (Ascaris suum) is present in the free-living nematode, Panagrellus redivivus (Nematoda, Rhabditida).

Available primary structural information suggests that the FMRFamide-related peptides (FaRPs) from parasitic and free-living nematodes are different, and that free-living forms may not represent appropriate models for the study of the neurochemistry of parasitic forms in the laboratory. However, here we report the isolation and unequivocal identification of AF2 (originally isolated from the parasite, Ascaris suum) from acidified alcoholic extracts of the free-living species, Panagrellus redivivus. While reverse-phase HPLC analysis of extracts revealed FMRFamide-immunoreactivity to be highly heterogeneous, AF2 was the predominant FMRFamide-immunoreactive peptide present (at least 26 pmol/g wet weight of worms). This peptide was also the major immunoreactant identified by an antiserum raised to the conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP), which has been used previously to isolate neuropeptide F (NPF). These observations were confirmed by radioimmunoassay and chromatographic fractionation of an acidified alcoholic extract of A. suum heads. The FMRFamide-related peptides present in a nematode extract may be highly dependent on the extraction medium employed, and these data would suggest that this complement of neuropeptides may not be as different between parasitic and free-living nematodes as initial studies have suggested. Finally, all of the evidence suggests that NPF is not present in nematodes and that the PP-immunoreactant previously demonstrated immunochemically is probably AF2.