Fluorescence fingerprints of Eisenia fetida and Eisenia andrei6

Photochemistry and Photobiology, Dec 2003 by Albani, J R, Demuynck, S, Grumiaux, F, Lepretre, A

Absorbance data were obtained with a Varian DMS-100 S spectrophotometer using 1 cm pathlength cuvettes.

Fluorescence spectra were recorded with a Perkin-Elmer LS-5B spectrofluorometer. The bandwidths used for the excitation and the emission were 5 nm. The quartz cuvettes had optical pathlengths equal to 1 and 0.4 cm for the emission and excitation wavelengths, respectively. Fluorescence spectra were corrected for the background intensities of the buffer solution. Corrections for the inner-filter effect were not necessary because the absorptions were nonsignificant at the excitation and emission wavelengths.

All experiments were performed in 10 mM Tris-HCl buffer, pH 8.6.

RESULTS AND DISCUSSION

Figure 2 displays the fluorescence emission spectra of coelomic fluids of E. andrei and E. fetida (spectra a and b, respectively). We notice that the fluorescence occurs only from E. andrei. The fluorescence observed from E. fetida is almost nonexistent, and in most of the earthworms studied, it was identical to the diffusion of the buffer. Thus, E. andrei and E. fetida are physiologically and metabolically different from each other because the coelomic fluid of E. andrei contains a fluorescent substance that is absent within the coelomic fluid of E. fetida.

The fluorescence excitation spectrum of a fluorophore characterizes the electron distribution of the molecule in the ground state. Thus, it helps to identify the structure or the nature (or both) of the emitting molecule. The fluorescence excitation spectrum recorded on the coelomic fluid of E. andrei shows that the fluorophore absorbs at a peak equal to 314 nm at pH 8 (Fig. 3). Fluorescence properties of the emitting fluorophore are characteristic of the 4-methylumbelliferyl [beta]-D-glucoronide (MUGlcU) (15). The absence of fluorescence in E. fetida indicates that the metabolism of this substrate is inhibited by one or more enzymes present only in the coelomic fluid of E. fetida.

TNS is a fluorescent probe that does not fluoresce when dissolved in a polar medium such as water but is highly fluorescent in a solvent of low polarity or when bound to a protein or to a membrane (16-18). Excitation at 280 nm of the coelomic fluid yields a fluorescence emission at 333 nm, characteristic of dissolved protein (data not shown). Addition of TNS induces a decrease in the fluorescence emission of the proteins and an increase of TNS fluorescence indicating an interaction between the extrinsic fluorophore and the proteins.

In presence of coelomic fluid, TNS shows a significant fluorescence. However, the position of the peak varies with the origin of the coelomic fluid. In fact, the emission peak of TNS is equal to 397 and 410 nm for E. andrei and E. fetida, respectively (Fig. 4). This difference is the result of the contribution of the emission of the coelomic fluid to the recorded emission spectrum. In fact, because there is no fluorescence occurring from the coelomic fluid of E. fetida, the observed emission in presence of TNS is from the extrinsic fluorophore only. Meanwhile, the spectrum recorded for E. andrei is the result of the contribution of both TNS and the natural fluorophore present in the coelomic fluid. Experiments with TNS confirm the fact that coelomic fluids of E. andrei and E. fetida differ, i.e. the metabolism of the two earthworms is not the same.