Fluorescence fingerprints of Eisenia fetida and Eisenia andrei6

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

ABSTRACT

We describe a fluorescent method that allows to differentiate the worms Eisenia fetida and Eisenia andrei. In fact, the coelomic fluid of E. andrei displays specific fluorescence absent in that of E. fetida. The two species do not metabolize the same types of molecules and thus can be differentiated at the molecular level. Each species has specific fluorescence fingerprints.

Abbreviations: mUGIcU, 4-methylumbelliferyl b-D-glucoronide; TNS: 2-p-toluidinyl-naphthalene-6-sulfonate.

INTRODUCTION

Performing an ecological and environmental diagnosis of an ecosystem supposes that one applies consistent methodologies on a population sensitive to modifications that occur within the environment. Among these methodologies, one can mention the diagnosis at the cellular or individual level and the analysis of the functioning of the ecosystem. All the approaches and tests used rest on the choice of the right species. Earthworms are the most important biotic components in the soil, and thus they are commonly used in studies of toxicity.

Eisenia andrei and Eisenia fetida are recommended by the Organization for Economic Cooperation and Development for tests of acute and subacute toxicity of soil. These worms are cultured easily in the laboratory (1), and an extensive database on effects of all classes of chemical on these two species is also available (2). These worms feed at the surface of the soil and thus reflect the impact of the recent contributions of soil pollutants. The two species display similar body length and segment number as well as show resemblance in the shape of the clitellum and tubercula pubertatis. They can be distinguished in the adult form because of their gross morphology, mainly their pigmentation (Fig. 1). The taxonomic status of Eisenia andrei Bouche 1972 and Eisenia fetida (Savigny, 1826) was confirmed by Jaenike (3) using electrophoretic techniques. Until recently they have been considered usually as subspecies according to their different body pigmentation. Andre (4) described Eisenia fetida form typica, with a characteristic stripped pattern and Eisenia fetida form unicolor with a uniform reddish colour. Bouche (5) considered that the term unicolor had a low systematic value, and thus designated these forms Eisenia fetida fetida and Eisenia fetida andrei. Andre was the first to demonstrate the specific status of these two forms by recording signs of reproductive isolation between them, but he did not give them the status of separate species. This status was given later, after biochemical characterizations of these species (3,6-8). Although they have been distinguished at the molecular level, they are still used indifferently in ecotoxicological studies, according to the norm ISO/TC 190/SC4 (9).

Recently, nuclear magnetic resonance studies performed on tissue extracts and on the coelomic fluid of E. andrei and E. fetida allowed to distinguish the two species (10). In general, spectroscopic methods such as fluorescence are sensitive to the structure and dynamics of molecules (11-13). In the present study, we performed fluorescence studies on the coelomic liquid of E. andrei and E. fetida. Our results show that the coelomic fluid of E. andrei displays a characteristic fluorescence that is absent in the coelomic fluid of E. fetida. Our results indicate that the two species do not metabolize the same types of molecules and thus can be differentiated quickly and easily at the molecular and metabolic levels.

MATERIALS AND METHODS

The earthworms used for the experiments were adults with a well-developed clitellum. The two species used, i.e. E. andrei and E. fetida, were obtained from our own culture stocks. Worms were bred at an ambient temperature of 20 � 5�C, in the dark, on a vegetable mould with fresh cattle manure (previously frozen to kill any pathogen) as the food source. The moisture content was maintained around 35%. Because earthworms are hermaphrodites, no sexual differences were taken into account during the experiments. Worms were transferred for 24 h to petri dishes containing filter paper moistened with distilled water to depurate before extracting coelomic fluid. For collection of the coelomic fluid, earthworms were placed individually in petri dishes with 250 �L, of 10 mM Tris-HCl buffer, pH 8.6 (Sigma Chemical Co., St Louis, MO). Electrical excitations were produced by a 9 V dry cell for 30 s to induce the extrusion of coelomic medium, i.e. coelomic fluid and the coelomocytes, through pores in the integument. The coelomic medium was then centrifugea at 4�C for 30 min at 7500 g in an Eppendorf 5840 R centrifuge to separate coelomocytes and mucous secretions from clear coelomic fluid. The coelomic fluid was used freshly prepared for the experiments. Ten worms of each species were used in this study.

2-p-Toluidinyl-naphmalene-6-sulfonate (TNS) was from Sigma. Its concentration was determined spectrophotometrically using an extinction coefficient of 18.9 mM^sup -1^ cm^sup -1^ at 317 nm (14).