Biochemical diversity of betaines in earthworms

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Abstract

The ability to accumulate osmoprotectant compounds, such as betaines, is an important evolutionary feature in many organisms. This is particularly the case for organisms that live in variable environments, which may have fluctuations in moisture and salinity levels. There is, surprisingly, very little known about betaines in soil invertebrates in general, and there is almost no information about earthworms – a group that are important ‘ecosystem engineers’ and key indicators of soil health. Here, we describe a fast and reliable 1H–13C heteronuclear single quantum coherence (HSQC) 2D NMR approach for the metabolic profiling of a series of betaines and related metabolites in tissue extracts, and list 1H and 13C chemical shifts for the trimethylammonium signal for 23 such compounds. The analysis of ten different species from three different families (Lumbricidae, Megascolecidae and Glossoscolecidae) showed an unexpected diversity of betaines present in earthworms. In total ten betaines were identified, including hydroxyproline-betaine, proline-betaine, taurine-betaine, GABA-betaine and histidine-betaine, and a further eleven as-yet unassigned putative betaine metabolites detected. The findings clearly indicate a hitherto-unappreciated important role for betaine metabolism in earthworms.

Highlights

► We develop a method for rapid untargetted analysis of betaines. ► We profile betaines in a comparative study of ten earthworm species. ► Earthworms contain a surprisingly high number of different betaine metabolites. ► Earthworms contain betaines normally seen only in plants or marine animals.

Introduction

Betaines – trimethylammonium derivatives of amino acids and related compounds – are an important set of metabolites for many organisms, including microbes, algae, plants, and animals, including humans [1], [2], [3], [4]. They play a pivotal role in osmoprotection of cells and tissues to maintain cellular homeostasis, and to protect them against environmental stresses like high salinity and extreme temperatures. Additionally, they serve as a catabolic resource of methyl groups in different biochemical pathways (transmethylation).

Betaines are well studied in bacteria [5], plants and algae [1], [6], but less is known about their occurrence in many other species. This is true for invertebrates, and in particular terrestrial invertebrates. Earthworms are a classic example where fundamental knowledge about their occurrence and distribution is lacking. Earthworms can survive and flourish in soils with both high and low extremes of water stress [7]. Soil moisture is considered to be the primary factor limiting survival of different earthworm species [8], [9], therefore they must preserve an efficient osmoregulatory system. Surprisingly little is known about betaines and other trimethylammonium compounds in earthworms. Our knowledge of which compounds may be present and their natural distribution is far from complete, but for instance metabolic profiling approaches by 1H NMR have detected both glycine-betaine and choline in Lumbricus rubellus [10] and Eisenia fetida [11]. This lack of knowledge motivated us to study the landscape of betaine compounds in earthworms. Additionally, we aimed to develop a fast and reliable method for the detection of these compounds, which can be easily applied to other sample types.

Betaines are zwitterionic quaternary ammonium compounds and so are not trivial to analyze by common separation methods such as reversed phase HPLC, although methods have been developed using hydrophilic interaction liquid chromatography [12] or a pentafluorophenylpropyl stationary phase [13] to separate betaines. More time-consuming protocols, including previous derivatization of betaines, can also be applied to improve HPLC retention characteristics [14]. In contrast, nuclear magnetic resonance (NMR) spectroscopy can also be used for analysis of betaines, and does not require physical separation of metabolites [1]. Betaine metabolites give rise to singlet resonances from the trimethylammonium group; because these are based on nine protons, and there is no resonance splitting, these compounds have relatively low detection levels by 1H NMR. However, it is difficult to assign betaines in 1D spectra based on the trimethylammonium peak alone; unfortunately, these are often the only peaks easily detected in crude cell/tissue extracts (especially for low-concentration compounds) because the other compound resonances are obscured by overlapping signals from other metabolites. Either homonuclear or heteronuclear two-dimensional NMR experiments are required for confident assignment of betaine metabolites in a given sample; in particular, heteronuclear 1H–13C methods make advantage of the inherently wide chemical shift distribution of the 13C nucleus. However, these can be time consuming, especially if aiming to give high-resolution spectra across the full range of proton and carbon shifts found in tissue extracts. Fortunately, as already described, betaines possess a characteristic trimethylammonium resonance, and spectral acquisition times can be drastically reduced (while maintaining resolution in both dimensions) by concentrating on this spectral region.

We present here an analytical approach that provides both qualitative and quantitative data, by combining short 1D and 2D HSQC NMR acquisitions of spectra from tissue extracts. We also provide a reference database for a range of amino acid derived betaines to consistently identify as many as possible compounds in various samples, and apply this to characterize the betaine profiles in 10 different earthworm species.

Section snippets

Earthworm species

Lumbricus rubellus (Lumbricidae) worms were a gift from David Spurgeon (CEH Wallingford, UK). Aporrectodea chlorotica (Lumbricidae) worms were collected from field populations within the UK. Lumbricus terrestris, E. fetida, and Dendrobaena veneta (all Lumbricidae), were purchased from Blades Biological Ltd. (Edenbridge, UK). Four additional species from two families were collected in glasshouses with tropical climate and flora based in the Royal Botanic Gardens, Kew, London (UK). These were

Results

To assess the occurrence of betaines in earthworms we screened 10 different earthworm species by a 1D 1H NMR metabolic profiling approach and refined a common 2D HSQC NMR approach into a rapid and sensitive method for betaine detection. The species were taken from three earthworm families, Lumbricidae, Megascolecidae and Glossoscolecidae. The Lumbricidae is the only earthworm family indigenous to the UK, and so, in order to increase the phylogenetic diversity of our study, we collected tropical

Discussion

Our final analytical method was fast, relatively sensitive, and can detect a whole range of betaines, including novel or unassigned compounds. The final acquisition time was reduced to 25 min, but still gave comparable signal-to-noise ratio to a two-hour HSQC experiment that covered the full chemical shift range. In fact, by reducing the chemical shift range and number of increments still further (e.g. to 8 ppm and 16 increments), we could reduce the total acquisition time to around 3 min per

Acknowledgments

We thank Emma Sherlock (Natural History Museum, London, UK) for her invaluable help in identifying worm species; the Director and Trustees of the Royal Botanic Gardens, Kew, London, UK, for access for worm collection; David Spurgeon (CEH Wallingford, UK) for provision of L. rubellus worms; and Peter Kille (University of Cardiff, UK) for provision of A. gracilis. This study was supported by the Natural Environment Research Council (NERC), under grant NE/H009973/1.

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