Identification of new fluorophores in coelomic fluid of Eisenia andrei earthworms

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.


Introduction
Morphologically similar lumbricid species occupying the same ecological niche, Eisenia andrei and E. fetida, can be phenotypically differentiated by metabolic profiling of tissue extracts and/ or coelomic fluid [1].Fluorescence spectra of coelomic fluid of these species were for the first time used for taxonomic purposes by Albani et al. [2], and E. andrei specific fluorophore was suggested as 4-methylumbelliferyl β-D-glucoronide (MUGlcU).In fact, similar spectra have been shown for methanol solutions of 4-methyl umbelliferone, a member of coumarin family [3].
Specific fluorescence spectra with a peak of excitation at 310-320 nm and a peak of emission at 370-380 nm, were used by our group as one of taxonomic markers of E. andrei, and called the MUG, MUG-like, or the M fluorescence.However, during these studies the MUG/ MUG-like fluorescence spectra were detected not only in E. andrei but also in a few of E. fetida earthworms [4][5][6] that inspired us for studies on hybridisation between laboratory joined inter-specific pairs of M-positive E. andrei with M-negative E. fetida.The results revealed the existence of asymmetrical hybridization between these hermaphroditic species able to self-fertilization [7], with hypothetical M fluorophore inherited by E. andrei-derived hybrids and then transferred to some of their E. fetida offspring [8].Recently, interspecific hybrids between E. andrei and E. fetida have been found among field-samples earthworms from Scandinavian populations [9].Perhaps the hypothetical M-fluorophore might be one of the markers of interspecific gene flow, thus studies on its proper identification and characteristics are pertinent.
An aromatic metabolite, called SP-8203, was isolated from coelomic fluid of E. andrei, consisting two quinazoline-2,4-diones joined by an N-acetylspermine linker [11,12].Later, its presence in coelomic fluid of E. andrei has been confirmed [13], although in none of these studies its fluorescence properties were analyzed.
In the present research we decided to isolate the most abundant fluorophores of E. andrei and determine their molecular structure and spectral properties.

Materials and methods
Investigations were performed on progeny of the composting earthworm species Eisenia andrei received from the laboratory stocks of the University in Lille (France) and reared for generations in the laboratory of Institute of Zoology and Biomedical Research of the Jagiellonian University (Krakow, Poland).In Krakow worms were routinely kept in boxes with commercial soil at 17˚C, 12:12 LD, and fed with a mixed diet comprised of dried/boiled nettle (Urtica dioica) and dandelion leaves (Taraxacum officinalis), and boiled/dried tea leaves.Adult (clitellate) earthworms of similar body weights were used for present investigations.
One hundred of adult individuals of E. andrei (total weight 30.4 g) was divided into 4 groups (25 individuals each, biomass ca.7.5 g).Each group was immersed in 10 ml of PBS (Fresenius Kabi Poland, Warsaw) and shocked for 60 s with 4.8V constant current.The solutions were pooled and treated twice by freeze-thaw cycles in liquid nitrogen.After centrifugation (5000g x 10 min) the sediment was discarded, while the supernatants were lyophilized and finally dissolved in 10 ml of the HPLC solvent (0.1% formic acid/ acetonitrile, 82.5/17.5 v/v).
Fluoresce spectra were recorded on a Perkin Elmer LS55 spectrofluorimeter using 5/5 nm excitation/emission slits, using quartz cuvette with 10 mm optical pathway.

LR-MS measurements
Low resolution mass spectra were recorded after HPLC separation on quadrupole tandem mass spectrometer (HPLC-QQQ MS/MS, 6410 LC/MS, Agilent, Santa Clara, CA, USA).The ion source was electro-spray ionization in positive mode (+ESI), at capillary voltage 4 kV, gas temperature 350˚C, gas flow 12 L/min and nebuliser pressure 35 psi.Separation was performed on AscentisExpress C18 (3×150 mm, 2.7 μm; Supelco, Bellefonte, PA, USA) analytical column in isocratic mode at 0.5 ml/min, injection was 5 μl, the same solvent mixture was used as for semipreparative separation.

HR-MS measurements
Mass spectrometry analyses were performed using Synapt G2-S mass spectrometer (Waters) equipped with the electrospray ion source and quadrupole-Time-of-flight mass analyzer.Sample was dissolved in methanol and injected directly into the electrospray ion source.Methanol was used as a solvent with the flow rate 100 μl/min.The measurement was performed in positive ion mode with the resolving power of the TOF analyzer 20000 FWHM.The instrument worked with external calibration on sodium formate in the mass range of m/z = 50-2000.The lock spray spectrum of the leucine-enkephalin was generated by the lock spray source and correction was done for every spectrum.The exact mass measurements for all peaks were performed within 3 mDa mass error.The nitrogen was used as desolvation and cone gas and their flow values were set to 861 L/h and of 222 L/h respectively.The desolvation gas flow was and temparature 150˚C.The nebulizer gas pressure was set to 5.8 bar.The capillary voltage was set to 3.8 kV, and the sampling cone voltage and source offset were set to 20 V. The instrument was controlled and recorded data were processed using the MassLynx V4.1 software package (Waters).The quantity of compound 6, used for NMR studies was 7 mg.

Results
The HPLC chromatogram of coelomic fluid extract showed (Fig 1) that it contains a mixture of several fluorescent compounds, however, the fluorophore corresponding to peak 6 is predominant.The percent composition of the fluorophores was as follows: 11.9% (compound 1), 0.7% (2), 9.0% (3), 2.6% (4), 0.6% (5) and 75.2% (6).The spectral data of the fluorophores (Table 1, Figs 2 and 3) show that the compounds corresponding to peaks 1, 3, 4 and 6 in the chromatogram show the same excitation and emission maxima, suggesting that they have the same chromophore.For compounds 2 and 5, the emission maxima are at longer wavelengths but have the same excitation maxima at 310 nm (Table 1, Fig 2).This indicates that chromophores of these compounds are of different but similar structure to those of compounds 1, 3, 4 and 6.As minor components, the compounds 2, 4 and 5 were not further analyzed in the present studies. .This metabolite is identical to SP-8203 reported earlier in coelomic fluid of E. andrei [11,13].
To verify the fluorescence properties of the quinazoline-2,4-dione ring, synthetic n-butyl quinazoline-2,4-dione was prepared and its fluorescence properties turned out to be identical to those of compounds 1, 3, 4 and 6 (Table 1, Figs 2 and 3

Discussion
Fluorescence spectra measured on lysates of coelomic fluid retrieved from E. andrei, most hybrids between E. andrei and E. fetida, and a few specimens of the latter species [4][5][6]8] are similar to those of compounds 1, 3, 4, and 6 (Fig 3).The fluorophore corresponding to peak 6, dominating in coelomic fluid of E. andrei is identical to that described previously and called SP-8203 [11], which was also synthesized and tested pharmacologically [11,12,15].This metabolite has also been independently found in  coelomic fluid of E. andrei [13], although in none of these previous studies its fluorescent properties were reported.In the present study, we have identified also two new compounds for E. andrei, one corresponding to peak 1 in the HPLC chromatogram, which differs from the compound 6 by the lack of N-acetyl group and was not reported yet in any organisms, as well as a compound that was already identified in E. fetida [13] and regarded as species specific.
However our studies show that this compound occurs also in E. andrei in significant amounts.
The presented data indicate that in the coelomic fluid of E. andrei there are also other compounds sharing the same (compound 4) or similar (compounds 2 and 5) chromophore structure with compound 6.These compounds require further studies.
The question remains as to the metabolic origin and physiological function of the investigated compounds.These are most probably formed by condensation of spermine with quinozoline-2,4-dione by earthworms.The first potential substrate was identified in coelomic fluid of E. fetida [16], while quinazoline derivatives are widespread in nature, especially in plants [17,18].To be considered is also bacterial origin of the investigated compounds as almost all lumbricid earthworms harbor extracellular vertically transmitted species-specific bacterial symbionts of the genus Verminephrobacter localized in their excetory nephridia [19,20].The other question is about physiological function of these compounds in earthworms, which could be antimicrobial and cytotoxic.It is known that quinazolinones have broad pharmacological and biological activity, such as herbicide action, antioxidant, antimicrobial, anticancer, antitubercular activity and others [14,21].
Riboflavin (vitamin B 2 ), having fluorescence spectra quite different from those of presently investigated fluorophores, was originally identified in E. fetida [22] and then detected in coelomic fluid and/or chloragogenous tissue of several lumbricid species [23,24].This vitamin plays a pivotal role in immunity [24][25][26][27] and regenerative processes [28].After experimental expulsion of colomocyte-containing coelomic fluid, both coelomocytes (amoebocytes and chloragocyte-derived eleocytes) and riboflavin are almost completely lost and amount of riboflavin is restored slowly, during 5-7 weeks, in parallel with numbers of chloragocyte-derived eleocytes.In contrast, during such treatment, the UV-emitting fluorophores are only partly depleted and restored much faster, during two-three weeks.It indicates that, in contrast to riboflavin, eleocytes are not the main source of the presently investigated compounds, and might support hypothesis about symbiotic bacterial involvement in metabolic pathways leading to formation of E. andrei-specific fluorophore.
The results of NMR analysis of compound 6 (S1-S5 Figs) led to the structure shown in Fig 4.This structure is consistent with the HR-MS data that gave m/z = 535.2676,leading to the formula C 28 H 35 N 6 O 5 for the [M+H] + ion (EM = 535.2669,Δ = 0.7 mDa) ), confirming the structure of the fluorescing moiety of these molecules.The fragmentation low-resolution (LR) mass spectra of compound 6 (S6 Fig) gave the following ions with m/z = 146.2,175.3, 203.4,274.4,and 316.5 that correspond to the depicted fragments in Fig 4. The LC-MS analysis of compound 1, gave [M+H] + = 493.6 and fragments with m/z = 146.3,175.3, 203.3, 247.5, 274.5 and 291.5.As compared to fragments of compound 6, no peak at m/z = 316.5 was observed in this case (S6 Fig), suggesting the absence of N-acyl group in the

Fig 2 .
Fig 2. Excitation and emission spectra of E. andrei UV-emitting fluorophores and of n-butyl-quinazolinedione.Excitation was at 320 nm for emission spectra, while emission was set at 390 nm for excitation spectra.The peak at 360 nm for compounds 2 and 5 in the emission spectra originates from Raman scattering.https://doi.org/10.1371/journal.pone.0214757.g002