Asymmetrical hybridization and gene flow between Eisenia andrei and E. fetida lumbricid earthworms

Uniformly pigmented Eisenia andrei (Ea) and striped E. fetida (Ef) lumbricid earthworms are hermaphrodites capable of self-fertilization, cross-fertilization, and asymmetrical hybridization. The latter was detected by genotyping of F1 and F2 progeny of the controlled Ea+Ef pairs by species-specific sequences of maternal mitochondrial COI genes and maternal/paternal nuclear S28 rRNA genes. Among F1offspring there were self-fertilized Ea (aAA), Ef (fFF), and cross-fertilized fertile Ea-derived hybrids (aAF); the latter mated with Ea and gave new generation of Ea and hybrids, while mated with Ef gave Ea, Ef, Ea-derived hybrids and sterile Ef-derived hybrids (fFA). Coelomic fluid of Ea exhibits unique fluorescence spectra called here the M-fluorescence considered as a molecular biomarker of this species. Since similar fluorescence was detected also in some Ef (hypothetical hybrids?), the aim of present investigations was to identify the M-positive earthworms among families genotyped previously. It was assumed that factor/s responsible for metabolic pathways leading to production of undefined yet M-fluorophore might be encoded/controlled by alleles of hypothetical nuclear gene of Eisenia sp. segregating independently from species-specific S28 rRNA nuclear genes, where 'MM' or 'Mm' alleles determine M-positivity while 'mm' alleles determine M-negative phenotypes. Spectra of M-fluorescence were detected in all 10 Ea (aAAMM) and 19 Ea-derived hybrids (aAFMm), three of four Ef-derived hybrids (fFAMm) and one 'atypical' Ef (fFFMm) among 13 Ef earthworms. Among progeny of 'atypical' M-positive Ef (fFFMm) reappeared 'typical' M-negative Ef (fFFmm), confirming such hypothesis. Alternatively, the M-fluorescence might be dependent on unknown gene products of vertically-transmitted Ea-specific symbiotic bacteria sexually transferred to the Ef partner. Hypotheses of intrinsic and external origin of M-fluorescence might complement each other. The presence/absence of M-fluorophore does not correspond with body pigmentation patterns; Ef-characteristic banding appeared in posterior parts of hybrids body. In conclusion, Ea/Ef hybridization may serve for further studies on bi-directional gene flow

Riboflavin content in autofluorescent earthworm coelomocytes is species-specific.

We have recently shown that a large proproportion of earthworm coelomocytes exhibit strong autofluorescence in some species (Dendrobaena veneta, Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion spp.), while autofluorescent coelomocytes are very scarce in representatives of Lumbricus spp. and Aporrectodea spp. Riboflavin (vitamin B2) was identified as a major fluorophore in Eisenia jetida coelomocytes. The main aim of the present experiments was to quantify riboflavin content in autofluorescent coelomocytes (eleocytes) from several earthworm species through a combination of flow cytometric and spectrofluorometric measurements. Spectrofluorometry of coelomocyte lysates showed that riboflavin was non-detectable in the coelomocytes of Aporrectodea spp. and Lumbricus spp., but was a prominent constituent of lysates from species with autofluorescent eleocytes. In the latter case, riboflavin content was the highest in E. fetida, followed by Octolasion spp. > A. chlorotica > D. rubidus. The riboflavin content of coelomocytes correlates positively with eleocyte autofluorescence intensity measured by flow cytometry and visible with fluorescence microscopy

Searching for external sources of the riboflavin stored in earthworm eleocytes

Riboflavin (vitamin B2) is essential to maintain immune potency in animals and plants. So far is accepted that animals cannot synthesise riboflavin; they rely on plant-sourced diets and intestinal bacteria for their supplies. A unique feature of earthworm ‘hepatocyte-like’ chloragocytes and chloragocyte-derived eleocytes floating in celomic cavity is the storage of riboflavin within intracellular granules. The hypothesis was that vegetarian food-deprivation or antibiotic/antifungal treatment inhibits riboflavin accumulation in eleocytes of Eisenia andrei. The 7-week starvation inhibited worm body weight gain and worm reproduction but had insignificant effects on celomocytes, both amoebocytes and eleocytes, and eleocyte riboflavin accumulation. The 1 week or 3 week antibiotic exposure had insignificant effects on worm coelomocytes and riboflavin content. Thus, a vegetarian diet and intestinal bacteria are not the exclusive or perhaps even the main sources of eleocyte riboflavin. The role of endosymbionts in earthworm flavonoid economy warrants targeted investigation. Moreover, the possibility of horizontal transfer of riboflavin biosynthesis genes from bacteria/fungi to earthworm genomes cannot be neglected