Aromatic metabolites from the coelomic fluid of Eisenia earthworm species

Earthworms from the genus Eisenia express coelomic fluid when under severe stress. This coelomic fluid contains a complex mixture of small-molecule metabolites, including aromatic metabolites which are known to be species-specific, yet their actual identities remain unknown. We have aimed to characterize selected high-concentration coelomic fluid metabolites. The major aromatic compound in Eisenia veneta coelomic fluid is the rare metabolite a-nicotinamide riboside; and the major aromatic compound for Eisenia fetida is closely related to the (already characterized) metabolite of Eisenia andrei, which consists of two aromatic quinazoline-2,4-dione ring structures linked by N-acetylspermine. The biological function(s) of these metabolites in earthworms is unknown, but we hypothesize that they represent remnants of larger molecules, possibly bacterial in origin, that are recalcitrant to metabolism by earth-

The closely-related species Eisenia fetida and E. andrei can be distinguished by colour [1], but are not otherwise separated by standard keys [2]. They were widely accepted as separate species only following the demonstration of electrophoretic separation of different protein isoforms between them [3,4], and they do not produce fertile crosses [5]. The precise taxonomic status of these species is complex and may require revision [6], but that is not our current concern. Instead, we are interested in the functional differences that underlie the separation of these closely-related species at the molecular level. Both species exude a pungent yellow liquid on rough handling or stress, which we refer to as exuded coelomic fluid (ECF). The yellow colour comes from riboflavin, which is found partly in the fluid, and partly associated with the large number of coelomocytes that are also released; Albani et al. [7] reported riboflavin in E. andrei, and Koziol et al. [8] also observed it in E. fetida. However, ECF also contains a complex mixture of small-molecule metabolites; the Eisenia veneta ECF is dominated by organic acids, including the citric acid cycle intermediates fumarate, succinate, malate, and a-ketoglutarate [9].
The E. fetida ECF also contains organic bases such as spermidine and putrescine, and membrane components such as myo-and scylloinositol [10]. Earthworm coelomic fluid has long been of interest as a complex matrix with many biological functions, including immune defence [11]; but in the context of excretion and transport of small molecules, most interest has been in the excretory system of the nephridiopore, with little attention paid to the coelomic fluid metabolite composition [12]. The untargeted NMR analyses show that the coelomic fluid has a high metabolic complexity.
An earlier study reported that, although NMR-based metabolic profiling of tissue extracts showed essentially no differences between E. fetida and E. andrei, their ECF profiles were dramatically different, particularly in signals from unidentified aromatic metabolites [13]. The species E. veneta had further differences in the aromatic metabolites present. However, these metabolites were not identified. Here, we assign the principal aromatic compound from E. veneta, and provide further evidence regarding the principal aromatic compound from E. fetida, and show that they are uncommon and unexpected metabolites, with unknown function in earthworms.
We extracted a large amount of ECF by treating many E. fetida and E. andrei (approximately 2000 per species) with 10% NaCl solution (1 L), which causes rapid expulsion of ECF. The worms were obtained from Blades Biological (Edenbridge, UK), and were gently washed in tap water before extraction to remove the residual compost medium. The coelomocytes are also generally lysed by this treatment, but we added an additional extraction step of the cell and debris pellet (following centrifugation) with~4 vol of methanol. We then used a C18 solid-phase extraction (SPE) cartridge (Sigma-Aldrich, UK) to desalt and concentrate the aromatic metabolites, before fractionating the sample using HPLC, using a 10 Â 250 mm ACE C18-AQ column (Hichrom, UK), eluting with a gradient of 0.1% aqueous ammonia solution to 30% acetonitrile, with UV detection at A 254 . Individual chromatographic peaks were collected manually in glass vials and dried down before resuspending in solvents for NMR and/or MS analysis. High-resolution FT-ICR mass spectrometry was performed using an LTQ FT Ultra (Thermo Fisher Scientific, Bremen, Germany) equipped with a chipbased direct infusion nanoelectrospray ion source (Triversa, Advion Biosciences, Ithaca, NY) in positive mode.
We also treated a much smaller number of E. veneta, about 100, in the same way to obtain a series of purified metabolites.
For E. veneta (obtained from a culture maintained at CEH Wallingford), we obtained higher quality 2D NMR spectra than we had managed before. Based on the comparison of the spectra to data reported in the literature [14], we assigned the main E. venetaspecific aromatic metabolite as a-nicotinamide riboside; this had previously been incorrectly described as nicotinamide mononucleotide [9], i.e. the actual metabolite is the nucleoside and not the nucleotide, and the a rather than the more common b anomer.
For E. andrei, we collected an intensely coloured peak, which we confirmed to be riboflavin by NMR and MS comparison to an authentic standard. However, the largest peak belonged to a metabolite eluting at 26.5 min. E. andrei ECF contains an aromatic metabolite that has been previously referred to only as SP-8203; it consists of two quinazoline-2,4-diones joined by an N-acetylspermine linker [15]. However, the aromatic metabolite that is specific to E. andrei has 16 aromatic resonances [13], which would initially seem not to be compatible with SP-8203. We found that redissolving this compound in either water or in an aprotic solvent, DMSO-d 6 , gave entirely different spectra. Varying the proportions of water and DMSO led to a smooth shift in the pattern of the aromatic peaks observed between the two extremes ( Fig. 1A), which we ascribe to differences in the proportion of molecules being held in different conformations between the two solvents, with four stable and equimolar conformers in water, and two conformers, in roughly a 2:1 ratio, in DMSO.
We then acquired homo-and heteronuclear 2D NMR data, using the (simpler) DMSO solution, and conclude that the previously unassigned metabolite is indeed SP-8203. (The 1 H and 13 C shifts for this metabolite have not previously been reported in the open literature, and are given in online supplementary information.) Furthermore, a high-resolution mass spectrum of this fraction had a major peak with 535.2666 m/z, which compares well to the predicted formula of 535.2669 for the [MþH] þ ion.
The E. fetida-specific metabolite gave rise to a peak in the mass spectrum at 510.2709 m/z, which corresponds to a possible molecular formula (for [MþH] þ ) of C 27 H 36 N 5 O 5 (calculated mass 510.2711, mass error ¼ 0.43 ppm), representing a difference of þH and ÀC, ÀN, from SP-8203. This immediately suggests a possible related metabolite structure (Fig. 1B), although the actual structure remains to be confirmed unambiguously.
Why do Eisenia species contain these unusual aromatic metabolites? It has been suggested that some of these metabolites may have direct functional roles e for instance, riboflavin is found in a wide number of earthworm species, and may play a direct role in earthworm immunity [16]. The compound SP-8203 is highly pharmacologically potent towards a number of endpoints in mammalian cells [17,18], although as yet nothing is known about its activity in its native earthworm host. An alternative hypothesis is that these metabolites may be accumulated breakdown products. Animals cannot synthesize riboflavin, and so all the riboflavin in ECF must be microbial in origin [19]. Earthworm coelomocytes include phagocytic immune cells [20], and potentially riboflavin could accumulate in coelomocytes and coelomic fluid from phagocytosed bacteria. Free a-nicotinamide riboside has only been reported once previously to our knowledge, as an antifouling metabolite from a sponge [21]. However, a-NADH can probably be epimerized in vivo from b-NADH, and then enzymatically converted to a-NAD [22]. It is plausible that the earthworm enzymes can readily degrade microbial b-NAD (the normal form), but that when a-NAD is present, they can metabolize the adenosine nucleotide part, but a-nicotinamide riboside is left behind as a recalcitrant residue. Earthworms adapt their 'normal' biochemical enzymes for phase II metabolism of xenobiotics [23], which could also potentially explain the quinazoline-dione metabolites, SP-8203 (from E. andrei), and the related compound from E. fetida (C 27 H 35 N 5 O 6 ). Lumbricus terrestris has been previously reported to conjugate the pesticide cypermethrin with spermine [24], and it is easy to see how SP-8203 could be derived from spermine (or N-acetylspermine) conjugation of smaller units. Again, therefore, SP-8203 and the E. fetida metabolite could be produced from other endogenous metabolites, and may not represent functional compounds in earthworms. We acknowledge that it is still, of course, possible that these metabolites may be directly functional in worms; similar to the proposed role of riboflavin in immunity, for instance [16]. Future work is needed both to characterize the responses of these metabolites to external factors, such as infection or stress, as well as to test their function directly.
There remains to be explained the striking differences between Eisenia species e why is a-nicotinamide riboside found exclusively in E. veneta, and different unique quinazoline-dione metabolites found exclusively in E. andrei and E. fetida? We do not have an answer, beyond noting here that the differences may lie either in the earthworms' own enzymatic capabilities, or in the associated microbiomes of the different species. This raises interesting questions for biologists interested in evolution of functional differences between closely-related species [25]. Furthermore, a better understanding of the possible source of metabolites in ECF will benefit future studies of changes in these metabolites in response to toxic or other environmental stress [10,26].