Barbara Plytycz

Nagroda Nobla w dziedzinie medycyny lub fizjologii przypadła w roku 2023 biochemiczce Katalin Karokó i immunologowi Drew Weissmanowi za opracowanie sposobu modyfikacji nukleozydów budujących nić mRNA w taki sposób, by wprowadzenie jej do komórki nie wywoływało stanu zapalnego, lecz pozwalało na efektywną syntezę zakodowanego w nich białka. Stało się to podstawą do opracowania szczepionek typu mRNA przeciw SARS-SoV-2, które uratowały miliony istot ludzkich przed ciężką chorobą lub śmiercią z powodu COVID-19.

The lumbricid earthworms Eisenia andrei (Ea) and E. fetida (Ef) have been used as model organisms for studies on hybridization. Previously they have been identified by species specific sequences of the mitochondrial COI gene of maternal origin (‘a’ or ‘f’) and the nuclear 28S gene of maternal/paternal origin (‘A’ or ‘F’). In experimental crosses, these hermaphroditic species produce progeny of genotypes Ea (aAA), Ef (fFF) and hybrids (aAF and fFA) originating by self-fertilization or cross-fertilization. To facilitate studies on new aspects of the breeding biology and hybridization of earthworms, polymorphic microsatellite markers were developed based on 12 Ea and 12 Ef specimens and validated on DNA samples extracted from 24 genotyped specimens (aAA, fFF, aAF and fFA) from three laboratory-raised families and 10 of them were applied in the present study. The results indicate that microsatellite markers are valuable tools for tracking interspecific gene flow between these species.

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.

Lumbricid earthworms Eisenia andrei (Ea) and E. fetida (Ef) are simultaneous hermaphrodites with reciprocal insemination capable of self-fertilization while the existence of hybridization of these two species was still debatable. During the present investigation fertile hybrids of Ea and Ef were detected. Virgin specimens of Ea and Ef were laboratory crossed (Ea+Ef) and their progeny was doubly identified. 1 –identified by species-specific maternally derived haploid mitochondrial DNA sequences of the COI gene being either ‘a’ for worms hatched from Ea ova or ‘f’ for worms hatched from Ef ova. 2 –identified by the diploid maternal/paternal nuclear DNA sequences of 28s rRNA gene being either ‘AA’ for Ea, ‘FF’ for Ef, or AF/FA for their hybrids derived either from the ‘aA’ or ‘fF’ ova, respectively. Among offspring of Ea+Ef pairs in F1 generation there were mainly aAA and fFF earthworms resulted from the facilitated self-fertilization and some aAF hybrids from aA ova but none fFA hybrids from fF ova. In F2 generation resulting from aAF hybrids mated with aAA a new generations of aAA and aAF hybrids were noticed, while aAF hybrids mated with fFF gave fFF and both aAF and fFA hybrids. Hybrids intercrossed together produced plenty of cocoons but no hatchlings independently whether aAF+aAF or aAF+fFA were mated. These results indicated that Ea and Ef species, easy to maintain in laboratory and commonly used as convenient models in biomedicine and ecotoxicology, may also serve in studies on molecular basis of interspecific barriers and mechanisms of introgression and speciation. Hypothetically, their asymmetrical hybridization can be modified by some external factors.

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. J. Exp. Zool. 321A: 28–40, 2014.