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Identification of new fluorophores in coelomic fluid of Eisenia andrei earthworms.

ABSTRACT: Coelomic fluid of Eisenia andrei contains a number of UV-fluorescent compounds. In the present study we have found that four of these compounds showed identical fluorescence excitation and emission maxima at 310 nm and 364 nm, respectively, suggesting they share the same chromophore. NMR and HR-MS spectroscopy of the most abundant fluorophore reavealed that its molecule is composed of two quinazoline-2,4-dione rings connected by spermine linker. This compound was earlier indentified in Eisenia andrei as SP-8203. Moreover, we have identified the structure of the two other fluorophores, one differing from SP-8203 by the absence of N-acetyl group, the compound not reported in any other organisms before, and the other already found in E. fetida and regarded as species specific. However, our results indicate that this metabolite is also present in E. andrei in significant amounts. The possible origin and function of these new metabolites is discussed.

SUBMITTER: Kruk J

PROVIDER: S-EPMC6438515 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Identification of new fluorophores in coelomic fluid of Eisenia andrei earthworms.

Kruk Jerzy J Dziurka Michał M Płytycz Barbara B

PloS one 20190328 3

Coelomic fluid of Eisenia andrei contains a number of UV-fluorescent compounds. In the present study we have found that four of these compounds showed identical fluorescence excitation and emission maxima at 310 nm and 364 nm, respectively, suggesting they share the same chromophore. NMR and HR-MS spectroscopy of the most abundant fluorophore reavealed that its molecule is composed of two quinazoline-2,4-dione rings connected by spermine linker. This compound was earlier indentified in Eisenia a ...[more]

PMID: 30921437

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Project description: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.

Project description:Earthworms are not endowed with adaptive immunity and they are rely on the tools of innate immunity. Cells of the innate immune system utilize pattern recognition receptors, such as Toll-like receptors, to detect the pathogen-associated molecular patterns (PAMPs). The first earthworm TLR was isolated from Eisenia andrei earthworms (EaTLR), which belongs to the single cysteine cluster TLR (sccTLR). Here, we identified a new multiple cysteine cluster TLR (mccTLR) in E. andrei earthworms. Phylogenetic DNA analysis revealed that it has no variability within one earthworm as well as in the population. By screening of the tissue expression profile, the TLR was expressed primarily in earthworm seminal vesicles and receptacles suggesting a connection to sperm cells. Seminal vesicles are often heavily infected by gregarine parasites. As a sign of immune response, a strong melanization reaction is visible around parasites. Stimulation experiments with profilin from related parasite Toxoplasma gondii, led to the upregulation of mccEaTLR in the earthworm seminal vesicles. Also, profilin activated prophenoloxidase cascade, the efficient mechanism of innate immunity. However, its involvement in the NF-κB signaling was not proven. Further, we provide evidence that the antibiotics metronidazole and griseofulvin destroyed the developing spermatocytes. The observed decrease in the mccEaTLR mRNA levels after the antibiotic treatment of parasites is caused by the decline of sperm cells numbers rather than by diminution of the parasites. Since earthworms with extensively reduced parasite load had a similar amount of mccEaTLR mRNA, presumably, earthworm sperm cells have a certain level of mccEaTLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mccEaTLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mccEaTLR in seminal vesicles correlates with the expression of endothelial monocyte-activation polypeptide II. High-throughput sequencing of gregarine DNA from seminal vesicles of individual earthworms resulted in great diversity of the observed genotypes. Phylogenetically, all observed OTUs belong to the clade of earthworm gregarines suggesting host specificity. Overall, mccEaTLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.

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Identification of new fluorophores in coelomic fluid of Eisenia andrei earthworms.

Publications

Identification of new fluorophores in coelomic fluid of Eisenia andrei earthworms.

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