ABSTRACT
The fatty acid composition of three North West European isolates of Heterorhabditis sp. from different geographical origins, UK211 (England), HF85 (The Netherlands) and EU17 (Estonia) was assessed directly after harvest and, for UK211 and HF85, after 5 weeks storage in water at 20 degrees C. Lipid represented 34-43% of the dry weight of fresh nematodes. Of this, neutral lipid (NL) comprised from 70% (HF85) to over 90% (UK211, EU17). The fatty acid patterns were similar between the three isolates. Oleic (C18:In-9), palmitic (C16:0), and linoleic (C18:2n-6) acid predominated with 51, 13 and 12%, respectively in the total lipid (TL) of fresh nematodes (average for the three isolates). Levels of unsaturation (U.I.) of fresh nematodes were on average 110, 112, 113 and 152 for the TL, NL, phospholipid and free fatty acid fractions, respectively. EU17 had a slightly lower U.I than the other two strains, despite its more northern origin. Changes in fatty acid composition due to storage were most significant in the NL fraction. The U.I. for the NL fraction increased during storage, suggesting a preferential use of saturated fatty acids.
Subject(s)
Fatty Acids/analysis , Nematoda/chemistry , Animals , Environment, Controlled , Lipids/analysis , Phospholipids/analysis , Survival , TemperatureABSTRACT
During storage, non-feeding stages of entomopathogenic nematodes become visibly more transparent due to depletion of energy reserves. Optical density per unit area (OD per area) of infective juveniles of Steinernerna carpocapsae (All) and two Heterorhabditis isolates (UK211 and HF85) was measured with an image analysis system and compared with neutral lipid levels obtained by Oil Red O staining. Optical density (OD) measurements were compared with triglyceride levels of UK211 and HF85. Good correlations between OD per area and neutral lipids (0.90) and between OD and triglycerides (0.87) were found. Thus, OD reflects lipid levels and can be used as an indicator of lipid reserves in these nematodes. Heat-killing of nematodes had no significant effect on OD measurements, but length increased significantly. Storage in a triethanolamine in formaldehyde solution decreased the OD and OD/area by about 5% to 8%. An additional advantage of the image analysis method described is that repeated measurements can be performed on live nematodes. Key words: entomopathogenic nematode, Heterorhabditis, image analysis, neutral lipid, Oil Red O, optical density, Steinernema, triglyceride.
ABSTRACT
Scanning electron micrographs of the nematode-egg-parasitic fungus Paecilomyces lilacinus infecting eggs of the root-knot nematode Meloidogyne spp. suggested the involvement of lytic enzymes. When grown on a liquid mineral salts medium, supplemented with different substrates as the sole N- and C-source, the fungus produced an extracellular protease. Colloidal chitin, vitellin and intact eggs of the root-knot nematode Meloidogyne hapla induced proteolytic activity that was repressed by glucose. The protease was partially purified from the culture filtrate by affinity chromatography. It has a molecular mass of 33.5 kDa, a pH optimum of 10.3, a temperature optimum of 60 degrees C and an isoelectric point above pH 10.2. The enzyme was completely inhibited by PMSF. The amino acid sequence, as derived from the nucleotide sequence of a cDNA clone, had high homology with several subtilisin-like serine proteases. It was shown that the purified enzyme degrades vitellin. The protease quantitatively bound to nematode eggs, and eggs incubated with the purified protease eventually floated. Incubation of the purified protease with nematode eggs significantly influenced their development as demonstrated by time-lapse microscopy. Immature eggs were highly vulnerable to protease treatments, whereas those containing a juvenile were more resistant. In addition, hatched larvae were not visibly affected by the protease. It can be concluded that the serine protease might play a role in penetration of the fungus through the egg-shell of nematodes.
