Camp. Biochem. fhysiol. Vol. IOX, No. 3,pp. 509-511, 1993 0306~4492/93 56.00+0.00 Printed in Great Britain 0 1993 Pergamon Press Ltd

ANALYSIS OF CATECHOLAMINES IN TURBELLARIANS OF LAKE BAIKAL

KRISTER ERIKssoN,*t MARIA REUTER* and OLEG TIMOSHKIN~

*Department of Biology, Abe Akademi University, Biocity, Abe, SF-20520, Finland (Tel. 358 21-654060; Fax 358 21-654748); and $Limnological Institute, Siberian Division of the Russian Academy of Sciences,

Irkutsk, Russia

(Received 4 January 1993; accepted for publication 5 February 1993)

Abstract-l. The catecholamine content in six endemic turbcllarians of Lake Baikal was examined with high pressure liquid chromatography (HPLC).

2. The planarians Archicotylus sp., Baikalobia sp., Bdellocephala angarensis, Rimacephalus arecepta, Sorocelis nigrofasciata and the lecithoepitheliat Geocentrophora wagini were analysed.

3. Dopa and dopamine (DA) were detected in all species and noradrenaline was detected in all species except Archicotylus. DA dominates in all species.

4. Neither adrenaline nor the DA-metabolite DOPAC were detected.

INTRODUCTION

The most primitive type of nervous system that exhibits centralization and cephalization is found in members of the phylum Platyhelminthes, the flat- worms. In the evolution of nervous systems, the appearance of the various types of chemical neuro- transmission is of much theoretical interest.

In the free-living turbellarians, the catecholamines dopamine (DA) and noradrenaline (NA) have been detected histochemically. Furthermore, immuno- reactions to the indoleamine serotonin (S-HT) and to histamine have been detected (Franquinet and Cata- nia, 1979; Hauser and Koopowitz, 1987; Reuter and Palmberg, 1989; Wikgren et al., 1990).

Chromatographical methods have verified the presence of DA, NA, 5-HT (Welsh and King, 1970; Algeri et al., 1983) and melatonin (Morita et al.,

1987) in planarians of the genus Dugesia, and DA and its precursor 3,4_dihydroxyphenylalanine (dopa) in the microturbellarian Stenostomum leucops (Reuter and Eriksson, 1991).

Lake Baikal, which was formed 20-25 million years ago during the Miocenic era, is unique in many aspects. With a greatest depth of about 1700 m, it is the deepest lake in the world. There is a large array of endemic organisms in the lake, including seven platyhelminth orders. At present, sufficient taxonom- ical data are available on 14 genera and 37 species of planarians, with 13 of the the genera and all the species being endemic, and two genera and nine endemic species of Lecithoepitheliata (Porfirieva and Timoshkin, 1989).

These turbellarians can be expected to have under- gone a separate evolution and speciation during their

tAuthor to whom correspondence should be addressed.

isolation in the lake. There is no conclusive infor- mation about the identity of monoamines in these animals. Information about their neuroactive sub- stances is clearly of interest for the discussion of archaic characters of the nervous system.

The aim of this study was to analyse the catechol- amine content in six turbellarian species from Lake Baikal with high pressure liquid chromatography (HPLC).

We have studied representatives from the two orders Seriata (triclad planarians) and Lecithoepithe- liata (the prorhynchid Geocentrophora wugini). Rep- resentatives for all three phylogenetic lines of planarians in the lake (Porfirieva, 1977) are included in the study. Buikafobiu sp. belongs to the first, Archicotylus sp. to the second and Bdellocephala

angarensis, Rimacephaius arecepta and Sorocelis

nigrofasciata belong to the third phylogenetic line. The different species have different ecological habi-

tats. The planarians Archicotylus sp., Baikalobia sp. and B. angurensis live in shallow water, while the planarian S. nigrofasciuta and the lecithoepitheliatan G. wugini live at depths between 10 and 200m. Generally, the presence of many giant forms is one of the most interesting peculiarities of the fauna in Lake Baikal. The planarian R. arecepta, measuring up to 110 mm, is one of the largest freshwater planarians in the world, and it lives at depths between 50 and 880 m.

MATERIALS AND METHODS

The animals were either caught by hand in shallow water or collected with a bottom sledge from depths ranging from a few to 250 m by the authors.

Only the head regions from the large planarians B. angarensis and R. arecepta were used.

509

510 K. ERIKSSON et al.

The tissue was homogenized in 0.4 M perchloric acid containing 3,4-dihydroxybenzylamine as internal standard and frozen in liquid nitrogen for subsequent transportation to Finland. After centrifugation (lS,OOOg, 20 min) and filtration (0.45 pm) of the homogenate, the catechol derivatives were purified on alumina according to the method of Eriksson and Persson (1982).

The HPLC equipment comprised a Shimadzu SIL-9A autoinjector, a Shimadzu LC-7A HPLC- pump and a Bioanalytical Systems LC4B ampero- metric detector (+0.75 V). The column was a Waters Nova-Pak Cl8 column (150 x 3.9m.m., 4 pm spheres) operated at a flow rate of 1.5 ml min’

To confirm the results, two different mobile phases were used. Mobile phase A contained 10.5 g citric acid monohydrate (CA), 6.8 g potassium phosphate (PP), 30ml acetonitrile (AcN), 250 mg octane sulfonic acid sodium salt (OSA) and 200~1 10% EDTA 1-l water at pH 2.80, and mobile phase B contained 7.8 g CA, 5.1 g PP, 22.5 ml AcN, 530 mg OSA and 200~1 10% EDTA at pH 3.20. All chemicals were purchased from Sigma except EDTA, CA, PP and AcN, which were ob- tained from Merck.

RESULTS

Dopa and DA could be detected in all species, and NA was found in all species except Archicotylus. As can be seen in Table 1, DA is the dominating catecholamine. Not shown in the table is the absence of adrenaline and DOPAC in all species. In some cases, when no accurate value could be calculated due to the small amount of sample, the presence of a substance is indicated with a plus.

In B. angarensis and R. arecepta, the concen- trations of dopa and DA are lower than in G. wagini and S. nigrofasciata.

In S. nigrofasciata, a substance that could not be identified was detected. This substance gave a peak that could be interpreted as NA in a high concen- tration when mobile phase A was used (Fig. 1A). When mobile phase B was used, it was evident that this substance was not NA (Fig. 1B).

Table I. Catecholamines in different turbellarian species. The values are given as pg/g-’ wet weight and are corrected for the recovery. When no value could be calculated, the

presence is indicated with a plus

Species

Planaria Bdellocephala angorensis Sorocelis nigrofasciata Rimocephalus arecepta Baikalobia sp. Archicotylus sp.

Lecithoepitheliata Geocentrophora wgini

DOPA DA NA

0.06 0.72 0.02 I S6 2.36 0.07 0.33 0.81 0.09 + 0.45 + + + _

1.16 2.25 0.08

A s

0.2 nA

Fig. 1 A, B. Chromatograms obtained by HPLC. as de- scribed in Materials and Methods, on the Baikal planarian Sorocelis nigrofasciata. A. When mobile phase A was used, noradrenaline (NA) co-eluted with a substance, here named X, that could not be identified. B. Mobile phase B prolonged the retention time for NA, thereby separating it from X, and also prolonged the retention times for dopamine (DA) and the internal standard (IS) 3,4-dihydroxybenzylamine. The

retention times for X and dopa changed very little.

DISCUSSION

The results on Baikal planarians are in agreement with earlier studies on the planarian genus Dugesia, with DA dominating over a small but ubiquitous amount of NA (Welsh and King, 1970; Algeri et al., 1983). This is the first chemical analysis of catechol- amines in the taxon Lecithoepitheliata, and the re- sults resemble those obtained in the planarians.

The unidentified substance (X) in S. nigrofasciata binds to the alumina and may therefore be catechohc. Some flatworms contain a monoamine oxidase (MAO) with a high affinity for DA (Moreno and Barrett, 1979). However, the unknown substance is not DOPAC, which would be the expected result of DA degradation by MAO. The DA-metabolites 3- methoxytyramine and homovanillic acid do not bind to alumina and can therefore be counted out.

Mobile phase B contained less AcN and more OSA than mobile phase A, and should generally give longer retention times. This was true for all substances except dopa and X. Dopa is a cate- cholaminoacid that, in contrast to the catechol- amines, retains the carboxylic group. This gives it shorter retention times at higher pH, while the cat- echolamines are less affected by pH. The rise in pH from 2.8 in mobile phase A to 3.2 in the slower mobile phase B made the retention time of dopa unaffected. The unidentified substance behaved in a similar man- ner and may therefore be carboxylated.

Unusual catecholaminoacids, 5-OH-dopa and cys- teinyl-dopas, have been detected in coelenterates

Catecholamines in Lake Baikal turbellarians 511

(Carlberg, 1988) and the unidentified peak needs Dugesia gonocephala. Comp. Biochem. Physiol. 74C,

further research. 27-30.

The concentrations of dopa relative to DA differ Carlberg M. (1988) Catecholamines and chemically related

significantly. G. wagini and S. nigrofasciata, that amino acids in coelenterates. In Neurobiology of fnuert- ebrates, Symp. Biol. Hung. (Edited by Salanki J. and

show a high concentration of dopa, were homogen- S.-Rozsa K.), Vol. 36, pp. 25535. Akadtmiai Kiado,

ized whole, while only the head regions of R. arecepta Budapest.

and B. angarensis were used. Dopa takes part as a Eriksson B.-M. and Persson B.-A. (1982) Determination of

component in the hardening of egg shells in many catecholamines in rat heart tissue and plasma samples by liquid chromatography with electrochemical detection.

flatworms (Ishida and Teshirogi, 1986, and references J. Chromatogr. 228, 1433154.

therein). All species in this investigation reproduce Franquinet R.-and Catania R. (1979) Localization histo-

via eggs and the results suggest a similar role for dopa fluorimetrique et tude microspectrofluorimetrique de la

in Baikal turbellarians. serotonine et des catecholamines chez une planaire entire

Dopa, DA and NA have also been detected in the et en tours de regeneration. C. Hebd. Acad. Sci. Ser. D. Sci. Nat. 289. 339-342.

tapevvorm Diphyllobothrium dendriticum (Gustafsson and Eriksson, 1991; Eriksson et al. unpublished). Dopa and DA have been detected in the primitive microturbellarian Stenostomum leucops which be- longs to the taxon Catenulida close to the base of the flatworm evolutionary radiation (Reuter and Eriksson, 1991).

The tapeworms and planarians belong to the taxon Neoophora, which includes the most advanced groups of Platyhelminthes, and the widespread oc- curence of dopa, DA and NA in such distantly related flatworm taxa indicates important functional roles for these substances even at this evolutionary level. It also indicates that both the dopaminergic and noradrenergic systems may have already arisen in ancestors to the flatworms.

Acknowledgements-We wish to thank the Agneta och Carl-Erik Olin Foundation and the Magnus Ehrnrooth Foundation for financial support, and the Limnological Institute of the Russian Academy of Sciences, Irkutsk, for providing help and equipment for the sample collection.

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