Microanalytical investigation of nematodes

Abstract

The chemical characterization of individual free-living nematodes is a challenge due to their small size and correspondingly, their low dry mass (1–3μg). The concentrations of minor (S, P, Ca, K) and trace elements (Fe, Zn, Cu, Ni, and Mn) of individual specimens (Xiphinema Vuittenezi) were determined by total reflection X-ray fluorescence (TXRF) spectrometry following a two step sample preparation procedure. The mean concentrations of minor and trace elements changed in the range of 1–4 and 0.01–0.4ng/μg, respectively. The relative standard deviation varied between 9.4 and 25.3% which was influenced not only by the analytical procedure but also by the biological deviations among the eleven investigated specimens. The stress effect caused by CuSO4 treatment resulted in significant change in the nematodes only in their Zn content. In the lack of certified reference material, the TXRF method was checked by inductively coupled plasma mass spectrometry (ICP-MS) analyzing solutions of 100 digested nematodes. The carbon and nitrogen content of nematodes was also measured by total C/N analyzer and their ratio amounted to 4.78±0.31 which is lower than the values published for the bacterial or fungal feeding ones. The way of copper uptake was investigated by scanning electron microscopy (SEM). Nematode treated with copper-nitrate was pecked out at the mid-body using focused ion beam (FIB) technique. In order to preserve the cross-section to be investigated, platinum coating was applied prior to the FIB operation. Energy dispersive X-ray analysis was performed along the cross-section measuring the distribution of platinum, sulfur and copper. It has been found that the sulfur and copper distributions were fairly similar following the copper treatment, and these elements had the highest concentration in the cuticle.