The Society for Integrative and Comparative Biology
Pheromonal Communication in Nereids and the Likely Intervention by Petroleum Derived Pollutants1
1 Cardiff University, Cardiff School of Biosciences, P.O. Box 915, Cardiff CF10 3TL, UK
2 Carl-v.-Ossietzky University, Institute for Pure and Applied Chemistry, P.O. Box 2503, D26111 Oldenburg, Germany
3 University of Wales, Aberystwyth, Institute of Biological Sciences, Edwrad Llwyd Building, Aberystwyth SY23 3DA, UK
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Nereis succinea and Platynereis dumerilii (Annelida, Polychaeta) are broadcast spawners and reproduce semelparously. The final events in reproduction, swarming and spawning are co-ordinated by sex pheromones.
A water-soluble fraction of crude oil, the volatile fraction (C9—C16) of EKO FISK crude oil was found to induce release of gametes in male nereids at levels <0.3 ppm.
Using vacuum distillation, column chromatography, preparative GC and GC-MS analysis we showed that C5-alkylated benzenes were most potent in inducing sperm release, of those n-butyl-4-methylbenzene and 1,4-diethyl-2-methylbenzene were found to induce release of gametes at concentrations 
4 nM. This threshold is lower than those reported for natural pheromones (nereithione: 60 nM, uric acid: 600 nM) but higher than background levels of aromatic compounds of 0.05 nM and below.
Other oil fractions showed additional effects, blocking pheromone reception or narcotising and intoxicating animals. Part of these effects could be assigned to naphthalenes at levels down to approx. 320 nM. In the original mixtures, their action was modified or compensated by the presence of gamete release inducing alkylated benzenes. Other highly paralysing substances remained elusive.
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INTRODUCTION |
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The reproduction of many nereid species is the terminal event of their live cycle and is well co-ordinated in space and time (Olive and Garwood, 1983
). The species Platynereis dumerlii and Nereis succinea are both broadcast spawners that use environmental cues (temperature and lunar periodicity) for induction of metamorphoses into the mature heteronereid and timing of spawning and a bouquet of chemical signals for the co-ordination of the actual spawning behaviour (Hauenschildt, 1956; Kinne, 1954; Hardege et al., 1990, 1998).
The first chemical signal described as a sex pheromone in P. dumerilii and also in N. succinea was a simple ketone, 5-methyl-3-heptanone (5M3H, Zeeck et al., 1988, 1990; Hardege et al., 1991). A number of other volatile, low molecular mass compounds (e.g., methyldecane and octenones) were found in the coelomic fluid of these and other nereid species (Zeeck et al., 1991; Bartels-Hardege et al., 1996) and thought to play a role in their reproduction.
Although the exact role of these compounds in the reproduction of nereids remains uncertain to date, their presence gives rise to the hypothesis of interference from compounds derived from crude oil and their breakdown products. Such effects had been reported previously in lobster and other crustaceans (Atema, 1977; Takahashi and Kitteredge, 1973) but generally little is known about effects of pollutants derived from petroleum on chemical communication systems.
Investigations into the abundance of petroleum-derived hydrocarbons in the waters of the German Bight (Müller, 1988) demonstrated a permanent pollution of the seawater by petroleum-derived aliphatic and aromatic hydrocarbons and oxygenated derivatives at levels of ng/L. The findings were in accordance with those at other sites close to either busy shipping routes or major combustion sources (Gschwendt et al., 1982; Schwarzenbach et al., 1978). Permanent pollution of the marine environment by diffuse sources at levels that exceeded the reported thresholds for pheromone action of 5M3H (Zeeck et al., 1990) was proposed and a series of investigations were undertaken to assess the effects of compounds derived from crude oil on the reproduction of nereids using P. dumerilii and N. succinea as model organisms.
These demonstrated that petroleum derived pollutants impact the reproduction of both species at a sublethal level of approx. 0.3 ppm. The biological activity was found in a distillate fraction of the oil that included n-nonane to n-dodecane. Males of both species responded with release of gametes but females showed no responses (Beckmann et al., 1995).
Here we report the results of further studies that aimed to identify the components in petroleum that are responsible for release of gametes in male specimen of P. dumerilii and N. succinea.
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MATERIAL AND METHODS |
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Animals
Platynereis dumerilii
A culture of approx. 2,000 specimen, that originated from Arcachon, France, was maintained in the laboratory as described by Zeeck et al., 1988
, 1990. Adults were cultured in crystallisation dishes (500 ml seawater, 20 adults/dish) and fed a diet of fish feed (Tetra marin), Artemia salina and spinach. Adults were separated by gender at its first clear indication after start of metamorphosis, which occurred with a semi-lunar periodicity. Only animals that had left their tubes and started swarming were used for behavioural assays.
Nereis succinea
Swarming animals were caught in the summer months at the Isefjord, Denmark around the time of new moon and transported back to Oldenburg in cooled containers (<16°C). A temporary culture was established to secure supply of heteronereids in the winter months. Immature animals (approx. 200) were collected from the lower Weser intertidal north of Bremerhaven and kept in sediment (20 cm sand on 10 cm pebble) under a layer of seawater in a 600 liter container. Seawater (20
) was drained from below the sediment layer the majority of which was recycled (Fluval 303 pump) and part replaced at approx. 40 liter/day. Animals were kept initially at 12°C under a light regime of 15 hr light, 9 hr dark and were fed fresh Mytelus edulis. Metamorphosis was induced by increasing the water temperature to 20°C at a rate of 0.5°C/day (Hardege et al., 1990). Swarming heteronereids for bioassays were directly taken from the water layer. The system was restocked after cessation of swarming activity and subsequent reduction of water temperature back to 12°C.
Chemicals
Seawater from the North Sea in the area of Helgoland was collected by the crew of the Coast Guard vessel "Mellum." Seawater was pasteurised for the culture of P. dumerilii and diluted with de-ionised water to 20 for culture of N. succinea.
Oil samples derived from samples of "Angel EKO Fisk crude" (25.01.82) and "MT Alandia Bay EKO Fisk crude" (23.11.93).
All solvents were GC or HPLC grade or higher, chemicals at least analytical grade.
Bioassay
The behavioural assays on Platynereis dumirilii were carried out in 100 ml crystallisation dishes containing 40 ml 32 seawater. Tests on Nereis succinea were carried out in 200 ml dishes containing 100 ml of 20 seawater. A single already swarming specimen was introduced into the dish and the sample deposited into its swimming path with a pipette or microlitre syringe (Hamilton). 40 µl of distillates and fractions of column chromatography were applied in tests on N. succinea and 10 µl in tests on P. dumerilii. Volumes of trapped eluates of the GC were in the order of 2 to 10 µl for both species. A release of gamete was recorded when the specimen released a clearly visible amount of gametes after increasing its swimming velocity within 45 min after addition of the sample. The observation period accounted for the slow release of active compounds from the poorly water-soluble distillates and fractions of column chromatography (Beckmann et al., 1995). Animals that had not responded were transferred into fresh seawater and tested against coelomic fluid of females. No response was recorded only when the test with coelomic fluid resulted in a release of gametes.
Distillation
Crude oil samples were distilled once over a 60 cm vigreux column at reduced pressure at p = 18 mbar to gain a fraction with a range of boiling points (boiling range) from n-nonane to n-nonadecane. Rectification of the distillate using a Fischer-Spaltrohr-system under reduced pressure (18 mbar) and at 90% reflux ratio was used to obtain oil fractions with narrower boiling ranges from e.g., n-nonane to n-undecane or n-tetradecane to n-hexadecane.
Group separation
Column chromatography of distillates was carried out on 1.1 x 80 cm column filled with silica gel 60 (Merck Ltd, 70–230 MESH, conditioned for 4hr at 180°C, no deactivation) the column head was packed with 2.5 g anhydrous sodium sulphate. The column was filled with n-hexane and 100 µl of sample were applied onto the column. Aliphatic and aromatic hydrocarbons were eluted with n-hexane. Aliphatic and alicyclic hydrocarbons eluted in the first 130 ml followed by the aromatic hydrocarbons to 250 ml and collected in fractions of 5 ml. Nitrogen, sulphur and oxygen containing compounds were eluted with 100 ml methanol/dichloromethane (10/90, v/v) into a single fraction.
Gas chromatography
Preparative separations were carried out on a Packard 439 GC over a 30 m x 0.25 mm fused silica coated with 1 µm DB 5 (J&W scientific) fitted with 50 cm deactivated fused silica pre-column (0.25 mm I.D.) and detector split (y-piece leading into two 95 cm deactivated fused silica post columns). Samples (2 µl) were injected 3 times in cold on-column mode and developed using a linear temperature program starting at 80°C and heating at 1°C/min to 250°C final temperature. One port of the detector split was fitted to an FID and recorded the chromatogram. The end of the other port was submerged in acetone (nanograde, 300 µl) to collect effluent compounds according to the signals of detection.
Analyses of fractions for structural identity were carried out on Magnum GC-MS system (Finnigan, Varian 3400 GC coupled with ion trap detector, ITD 40). Sample (1 µl) were injected splitless at 250°C separated over 30 m x 0.25 mm fused silica capillary column with 0.25 µm DB 5-MS (J&W scientific) operated with helium at 12 psi using a linear temperature program starting at 50°C heating to 250°C at 1°C/min. The effluent was ionised in CI (iso-butane) or EI mode and spectra recorded in a mass range from 38–400 m/z; data were processed with Magnum V2.30 and spectra compared to NIST library entries.
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RESULTS |
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The behavioural assays of the fractions of the rectification showed that only oil fractions in the boiling range from n-nonane to n-heptadecane triggered any responses. Test animals reacted to these with release of gametes but also showed toxic effects, paralysis and repellent behaviour. Only one fraction was found that exclusively caused release of gametes in 80% of Nereis succinea (n = 20) and 100% of Platynereis dumerilii (n = 12). The GC-MS analysis showed that the fraction represented a boiling range from n-undecane to n-tridecane (C11–C13) and contained a high amount of alkylated benzenes.
The further separation of the C11–C13 fraction on silica gel yielded 12 fractions containing aliphatic and alicyclic, 24 containing aromatic and 1 containing nitrogen, sulphur and oxygen containing compounds (NSO fraction). Behavioral assays of these fractions demonstrated that only two of the aromatic fractions caused release of gametes in N. succinea (90%, n = 20) and P. dumerilii (60%, n = 10). Tests of the aliphatic fractions and the NSO fractions showed no effect on both species.
The further separation of one of the active aromatic fractions by GC yielded a total of 8 fractions, of which only two triggered a positive response. Release of gametes was observed in all tested N. succinea (n = 18) after exposure to 10 µl of sample and in 28 of 36 tested P. dumerilii after exposure to 2–5 µl. The GC-MS analysis of the gamete-release inducing fractions showed a narrow selection of potentially active compounds all of which were C5-alkylated benzenes. The GC separation did, however, only in one case (1,4-diethyl-2-methyl-benzene) succeed in isolation of a single compound. The identification of active compounds was consequently based largely on comparison of chromatographic and spectroscopic data from GC-MS measurements with existing data, e.g., NIST MS spectra library, recorded biological responses and only in few cases could refer to synthetic compounds.
The analysis resulted in 12 compounds that were potentially able to elicit release of gametes in N. succinea and P. dumerilii males. In the case of N. succinea it was not possible to further clarify the identity of these but in P. dumerilii the activity of n-butyl-4-methyl-benzene was confirmed by synthesis whereas the synthetic isomer n-butyl-2-methyl-benzene was found to increase swimming activity but not to induce release of gametes. 1,4-diethyl-2-methyl-benzene was sufficiently pure to be positively identified but no further proof for the activity of the remaining compounds was undertaken. Structures, names and thresholds of these compounds are listed in Table 1.
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Effects of fractions of higher boiling distillates (C13–C17) were further investigated on P. dumerilii males. The animals showed no responses to the aliphatic/alicyclic and NSO fraction from the silica gel separation of these fractions but exhibited distinct responses to the aromatic fraction. The incidence of responses appeared to follow the retention on silica gel. Induction of gamete release was highest (15 of 32) in the early eluting aromatic fractions (131–170 ml) compared to the medium (171–210 ml) and late (211–250 ml) eluting fractions (2 of 28, 2 of 23). A paralysing effect was observed with higher incidence (9 of 23) in late eluting fractions and to lesser extend in the medium and early eluting fractions (5 of 28, 1 of 32). GC-MS analysis of early eluting fractions detected predominantly alkylated benzenes with molar masses from 176 to 218 g/mol (C7–C10). Analysis of medium eluting fractions showed abundance of dihydro- and tetrahydro-naphthalenes, dihydro-indenes and their alkyl-derivatives. Late eluting aromatic fractions composed predominantly of bicyclic and polycyclic aromatic compounds, namely alkyl-napthalenes and alkyl-indenes. Medium and late eluting fractions also contained to a lesser degree higher homologues of alkylated benzenes (M > 218 g/mol, C10+).
Aromatic compounds from higher boiling distillate fraction were not investigated in more detail. Only methyl and ethyl naphthalenes, which were abundant in the late eluting aromatic fraction and also commercial available were investigated for their effects on the behaviour of P. dumerilii.
The results of the behavioural tests indicated that all compounds predominantly have a paralysing effect and, with the exception of 2-ethyl-naphthalene, increased swimming velocity. Release of gametes was observed with both 2-alkyl-naphthalenes with low incidence (Table 2).
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DISCUSSION |
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The study clearly showed that oil-derived pollutants cause release of gametes in males of the nereids Nereis succinea and Platynereis dumerilii and that the effect can be assigned to a small selection of C5-alkylated benzenes.
The study also revealed other effects, which also appeared to be caused by particular components of the oil. These were not followed in much detail and only methyl and ethyl-naphthalenes were investigated. They too caused release of gametes in male nereids but at much higher levels and with much lower incidence compared to the benzenes.
The results suggest that effects of oil-derived pollutants are structure dependent as only aromatic compounds caused any effects in the n-alkane range from C11 to C17, only a selection of a total of 51 possible isomeric C5-alkylated benzenes were found to unambiguously induce release of gametes in both species and the active isomers were not the same for both species. It is not possible to deduce any structure-activity relationship form the data but it is clear that the active C5-alkylated benzene isomers are with approx. 4 nmol/ L threshold level much more potent inducers of release of gametes than the natural pheromones cysteinyl-glutathione in N. succinea (60 nmol/L, Zeeck et al., 1998a) and uric acid in P. dumerilii (600 nmol/L, Zeeck et al., 1998b). Consequently oil pollution events can seriously impact reproduction of both species. C5-alkylated benzenes have not been reported in environmental samples from potentially chronic polluted sites at concentrations approaching 4 nmol/L (Müller, 1988; Gschwendt et al., 1982; Schwarzenbach et al., 1978) but will reach and exceed such levels in cases of acute pollution events (Reddy and Quinn, 1999). The potential for such a disturbance would also be limited to the reproductive season of the nereids. In northern Europe both species reproduce from June to end of September; the precise time of swarming is co-ordinated by lunar periodicity and occurs around new moon in Nereis succinea (Hardege et al., 1990) and first and third quarter of the lunar cycle in Platynereis dumerilii (Hauenschildt, 1956). These reproductive events, however, take place on limited spatial scales and close to the water surface. According to an estimate by Beckmann et al. (1995) 1 litre of EKO Fisk crude or Arabian light could contaminate 2 m3 of seawater or an area of 8 m2 to a depth of 0.25 m to levels high enough to induce release of gametes. Consequently even smaller locally restricted oil spills of only a few litres could be disastrous for a local nereid population.
Interferences of oil pollution with chemical communication systems have often been suggested (Atema et al., 1982) but only a few studies have been concerned with such effects. The chemoreception of Homarus americanus and Pachygrapsus crassipes is effectively disrupted by oil derived pollutants at similar concentrations as those reported for nereids (Atema, 1977; Takahashi and Kittredge, 1973). The authors discuss the possibility that such interference could result in a limited ability to find food or mates or to detect and escape from predators. Similarly the chemoreception of motile bacteria is affected by petroleum-derived hydrocarbons blocking their ability to detect potential food sources (Mitchell et al., 1972).
Chemical communication is widely used in the marine environment (Chivers and Smith, 1998; Hay, 1996) and there is little reason to believe that other chemical communication systems should remain unaffected by oil-derived compounds. Acute and persistent pollution with oil-derived compounds may well influence a significant number of life cycle events in a variety of species and consequently the ecology of exposed habitats. The example of nereids demonstrates the potential impact of such interference in a single event on the population dynamics of the species and also that only few compounds were responsible for the observed effects. The study also showed that different components exert distinct effects and can selectively influence single traits at very low levels. Only further studies on nereids and other organisms will allow a better evaluation of the actual impact and chemistry of acute and persistent oil pollution on chemical communication in marine environments.
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ACKNOWLEDGMENTS |
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The authors wish to thank Dr. J.D. Hardege and H.D. Bartels-Hardege for advice on nereids, Dr A. Stief and Ms A. Brakenhoff for advice and support in nereid cultures, Mr K.H. Plate for advice on MS matters and the crew of the MS ‘Mellum’ for supplies of seawater.
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FOOTNOTES |
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1 From the Symposium EcoPhysiology and Conservation: The Contribution of Endocrinology and Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Louisiana.
2 E-mail: MullerCT{at}cf.ac.uk
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References |
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Atema, J. 1977. Effects of oil on lobster. Oceanus, 20:63-73.
Atema, J., E. B. Karnofsky, S. Olszko-Szuts, and B. Bryant. 1982. Project Summary: Sublethal effects of No 2 fuel oil on lobsters' behavior and chemoreception. Research and development, EPA-600/S3–82–013.
Bartels-Hardege, H. D., J. D. Hardege, E. Zeeck, C. T. Müller, B. L. Wu, and M. Y. Zhu. 1996. Sex pheromones in marine polychaetes V: A biologically active volatile compound from the coelomic fluid of female Nereis (Neanthes) japonica (Annelida, Polychaeta). JEMBE, 201:275-284.
Beckmann, M., J. D. Hardege, and E. Zeeck. 1995. Effects of the volatile fraction of crude oil on spawning behaviour of nereids (Annelida, Polychaeta). Mar. Env. Res, 40:267-276.[CrossRef]
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Zeeck, E., J. D. Hardege, H. D. Bartels-Hardege, A. Willig, and G. Wesselmann. 1991. Sex pheromones in a marine polychaete: Biologically active compounds from female Platynereis dumerilii. J. Exp. Zool, 260:93-98.[CrossRef]
Zeeck, E., C. T. Müller, M. Beckmann, J. D. Hardege, U. Papke, V. Sinnwell, F. Schroeder, and W. Francke. 1998a. Cysteine-glutathione disulfide, the sperm release pheromone of the marine polychaete Nereis succinea (Annelida, Polychaeta). Chemoecology, 8:33-38.[CrossRef]
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