Coelomocyte-derived fluorescence and DNA markers of composting earthworm species

Abstract

Supravital species identification of morphologically similar syntopic earthworms inhabiting dung and compost heaps or those from commercial cultures is difficult. The aim of the studies was to find out non-invasive species-specific markers for proper segregation of earthworm species from a dense mixed colony of waste decomposers. Worms were segregated according to external characteristics into Eisenia andrei, Eisenia fetida, and Dendrobaena veneta, and left for reproduction and analysis of non-invasively retrieved coelomocyte-containing coelomic fluid and/or species-specific partial sequences of cytochrome c oxidase subunit I (COI) gene in DNA extracted from amputated tail tips of adults and their offspring. Flow cytometric analysis of coelomocyte samples revealed that amount of nuclear DNA increases in order D. veneta ≪ E. andrei < E. fetida, and intensity of eleocyte-derived fluorescence is lower in D. veneta than in Eisenia spp. Spectrofluorimetry of coelomocyte lysates revealed that the amount of eleocyte-stored riboflavin is significantly lower in coelomocyte lysates from D. veneta than from Eisenia spp., and the emission peak of X-fluorophore is much more distinct in D. veneta than in Eisenia spp. Coelomic fluid of E. andrei exhibits a very distinct spectra of MUG fluorophore which are absent in D. veneta and in the majority of E. fetida, while some E. fetida possess MUG-like fluorophore. Sequences of the COI gene in the DNA of the worms from the mixed colony and their offspring confirmed species identity. In conclusion, species-specific coelomocyte-derived markers may be a useful complement to morphological and DNA-based taxonomy during studies on syntopic earthworms.

Differential Time Course of Restoration of Experimentally Depleted Coelomocytes and Fluorophores in the Earthworm <I>Eisenia andrei</I>

“…Eisenia andrei (Lumbricidae, Annelida) derived from a laboratory culture at the Lille University (France), their taxonomic identity having been confirmed by barcoding and several molecular markers (RORAT et al 2014 …”

“…Coelomic fluid with suspended coelomocytes was expelled through the dorsal pores of electrostimulated earthworms, as described previously (RORAT et al 2014). Coelomic fluid extrusion was always performed on depurated earthworms kept overnight on wet filter papers, and then gently washed/dried.…”

“…Coelomic fluid extrusion was always performed on depurated earthworms kept overnight on wet filter papers, and then gently washed/dried. Then the weighed earthworms were individually placed in Petri dishes containing 3 ml of extrusion fluid (phosphate-buffered saline, PBS) supplemented with 2.5 g/l ethylenediamine tetra-acetic acid (EDTA; Sigma-Aldrich) to avoid cell clumping and electrostimulated for 30 sec with an electric current (4.5V) as described previously (RORAT et al 2014). 2ml samples of the extruded coelomocyte suspensions were used for spectrofluorimetric analysis, while 1 ml was used for microscopic analyses and cell counts in haemocytometer.…”

“…In our experience, MUG was found to be present in every individual of E. andrei that we have investigated. However, qualitatively similar fluorescence spectra, albeit of much lower intensity, were recorded in some specimens of E. fetida (RORAT et al 2014). Whilst the low intensity 'noise' of MUG in a minority cohort of E. fetida does not invalidate the use of this molecule as a biomarker for distinguishing E. andrei from the other species, it is important to establish how the standard technique of electrostimulation for coelomic fluid sampling affects MUG levels in E. andrei.…”

“…Since these species are much favoured for ecotoxicological investigations, where the possibility of inter-species differences in sensitivity must be embraced, rapid and reliable high-throughput means of identifying individual worms is an important prerequisite. For this reason we have developed non-invasive markers, verified by molecular-genetic probes, to distinguish the three composting species (RORAT et al 2014). Among the potentially useful markers is a fluorophore present in coelomic fluid of E. andrei, that is quite distinct from riboflavin.…”