Biochemical composition and sediment temperature in relation to the reproductive cycle in the Lugworm arenicola marina

Netherlands Journal of Sea Research 19 (2): 111-118 (1985)

BIOCHEMICAL COMPOSITION AND SEDIMENT TEMPERATURE IN RELATION TO THE REPRODUCTIVE CYCLE IN THE LUGWORM ARENICOLA MARINA

E. MAYES and D.I.D. HOWlE Zoology Department, Trinity COllege, Dublin 2, Ireland

ABSTRACT Sediment temperature, and the biochemical composition of lugworm tissues, have been studied in relation to the reproductive cycle. Frequency distributions of oocyte diameter were used as an index of reproductive maturity. There is some evidence that the annual cycle of sedi. ment temperature has a role in controlling events in the reproductive cycle. Fluctuations in biochemical composition are due chiefly to the storage and utilisation of reserve materials in the reproductive process. Protein and lipid in the gut tissue are the main reserves used in gametoo genesis.

ual worms in a population (see OLWE, 1980). In this study, hourly records of sediment temperature were made, and the annual cycle of temperature considered in relation to the reproductive cycle. The biochemical changes in somatic tissues associated with reproduction are poorly known in polychaete species. Some observations on the biochemical composition of lugworm tissues have been published (DALES, 1957, 1958; DE VOOYS, 1975), but these were not related to the precise stage of reproductive development. In this study, the biochemical composition of lugworm tissues has been examinated for evidence of nutrient storage and utilization in the reproductive process, using oocyte diameter as an index of reproductive maturity.

1. INTRODUCTION The lugworm Arenicola m a r i n a L. is a polytelic species, breeding several times per life span but with discrete reproduction and frequently with only one reproductive crisis per year (OLWE & CLARK, 1978; HOWlE, 1984). After shedding from the gonad, gamete development is solitary, and takes place over a period of several months while the gametes are suspended in the coelomic fluid. Some aspects of the reproductive cycle are under endocrine control. In male worms the total number of gametes produced is regulated by a negative feedback mechanism acting on the gonad (OLIVE,1972) and in both sexes final ripening of the gametes is brought about by a maturation hormone (HOWlE, 1963, 1966; MEIJER & DURCHON,1977). The function of these endocrine controls appears to be the synchronisation of the gamete population within individual worms prior to spawning. However, given the annual periodicity of reproduction in the lugworm, there must be some input of exogenous information which brings about synchronisation among the individ-

Acknowledgements.--The authors are grateful to Prof. J.N.R. Grainger for help in setting up the temperature recording apparatus and for his assistance in the analysis of the data. Mr. F. Walker's assistance in maintaining the apparatus is also acknowledged. 2. METHODS Lugworms were collected at Booterstown, Co. Dublin, from an area of shore exposed for approximately 4 hours at low tide. They were transported to the laboratory in sea water, placed in clean sea water in individual plastic containers in a cool room, and left overnight to allow the guts to empty. Two collections of worms were made within a few days of each other at least once a month. The first collection was used for detailed assessment of the stage of reproductive development of the worm population, the second collection was used for biochemical analysis. Worms were blotted dry and weighed to the nearest 0.1 g. Samples of coelomic fluid (-0.1

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E. MAYES & D.I.D HOWIE

ml) were withdrawn by syringe and examined under a microscope for the presence of gametes. The bulk of the population could be sexed throughout the year. In the case of female worms, maximum oocyte diameter was measured using an eyepiece micrometer. Percentage frequency distributions of oocyte diameter were calculated for each individual, and as distributions were usually non-normal, median rather than mean values were calculated. A random sample of 50 oocytes was measured for each individual. Worms were assigned to groups of a similar degree of maturity on the basis of frequency distribution of oocyte diameter as follows: >50% of oocytes less than 50 #m diameter (Group I), >50% of oocytes between 50 and 150 #m diameter (Group II), and > 5 0 % of oocytes greater than 150 #m diameter (Group III). These groupings correspond, respectively, to the previtellogenic, vitellogenic and post-vitellogenic phases of oocyte growth, as described by RASHAH & HOWlE (1982). Having assessed the degree of maturity in the population from the first collection, worms for use in biochemical analysis were assigned to groups in the same way, on the basis of random measurements of 15 oocytes. Individuals which could not be grouped because no size category contained more than 50% of the oocytes were omitted, as were damaged worms. No formal measurements of the stage of development of gametes were carried out in male worms. However, individuals of approximately similar development were grouped together on the basis of criteria such as the presence or absence of mature sperm in morulae in the coelomic fluid. Worms for use in biochemical analysis were sacrificed not later than the second day following collection. Pooled samples of coelomic fluid, body wall (including musculature) and gut were analysed separately for each sex. Tails were discarded. These samples were derived from an average of 12 worms. Coelomic fluid samples were withdrawn by syringe, taking care to avoid contamination with blood. Samples were kept on ice, centrifuged at 2.800 r.p.m, for 25 minutes, and the supernatant coelomic fluid stored at - 17°C. The worms were then dissected, and the body wall separated from the trunk gut, and kept on ice. Tissues were rinsed in three changes of distilled water to remove coelomic contents, homogenized and dried overnight at 80°C. When dry, tissues were ground with a pestle and mortar and stored in a dessicator.

Biochemical analysis.--Three replicates were analysed in each determination, averages calculated, and results expressed as mg.g -1 dry weight. Protein.--Protein levels in both tissues and coeIomic fluid were determined by a Kjeldahl estimation of total nitrogen. Digestion of samples was according to ALLEN (1974), and colorimetric determination was by SCHEINEB'S (1976) method. On one occasion, samples of coelomic fluid from individual female worms were analysed by determination of nitrate. In this method, organic nitrogen and ammonia are oxidized to nitrate by alkaline persulphate digestion. Subsequent acidification facilitates direct measurement of nitrate by U.V. spectrophotometry (AMERICAN PUBLIC HEALTH ASSOCIATION, 1975; SOLORZANO & SHARP, 1980). Lipid.--Total lipid was estimated gravimetrically by the method of BLIGH & DYER (1959). Values were approximate as no account was taken of the effect of oxidation during drying. Glycogen.--Glycogen was determined according to DE ZWAAN & ZANDEE (1972) with some modification as dried tissue was used. Samples of the order of 30 mg were digested in 10 ml of 30% KOH for 2 hours at 50°C. Digests were made up to 50 ml with distilled water. Three ml samples were taken for colorimetric determination, which was according to DE ZWAAN & ZANDEE save in one respect. Samples plus Anthrone reagent were heated for 5 minutes at 100°C as this was found to give a more stable colour reaction. Anthrone reagent was prepared by adding 200 mg Anthrone per 100 ml diluted Analar sulphuric acid (76 ml acid: 30 ml distilled water). Oyster glycogen (Sigma Type II) was used as a standard. Temperature record.--Sediment temperature was measured by thermistors and recorded automatically by a Microdata logger model 200, sited approximately 2.5 km from the area where the lugworm samples were collected, but at a similar tidal level (i.e., exposed for approximately four hours at low tide). Thermistor probes were attached to a piece of angle iron and buried in the sand at depths of 5 and 25 cm. These depths were chosen as delimiting the range of depth in the sediment occupied by lugworms in the course of their activity cycles of feeding, irrigation and defecation, throughout the year in Dub-

BIOCHEMICAL COMPOSITION IN ARENICOLA

lin Bay. The channels were read every hour, and the data stored on magnetic tape. There were some failures in the record due to accidental cutting of the leads on the land end, w h i c h particularly affected the 25 cm record during the winter of 1980/81. Comparative air temperatures were screen temperatures measured at the synoptic meteorological station at Dublin Airport• 3. RESULTS 3•1.

SEDIMENT TEMPERATURE AND THE REPRODUCTIVE CYCLE

Sediment temperatures at depths of 5 and 25 cm were averaged for the three hours closest to high, and to low tide, over periods of a fortnight• There was little difference between mean temperatures at high and low tide, although at 5 cm depth standard deviations of the mean were greater at low than at high tide. Standard deviations were considerably smaller at 25 cm than at

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5 cm. At both sediment depths, temperature began to rise at the beginning of March (Fig. la) and increased steadily to reach maximum values in August. The peak temperature of 17.5°C at 5 cm occurred in early August, while at 25 cm the peak temperature of 19.2°C occurred slightly later in the second half of August• From the end of April on, higher mean temperatures were seen at 25 cm than at 5 cm depth• During the autumn, temperature loss occurred more rapidly at 5 than at 25 cm. Fig. 2 shows the relationship between air and sediment temperatures during selected 3 day periods at different times of the year. Fluctuations in sediment temperature appear to be influenced primarily by air temperature, with tidal cycle showing a subsidiary effect (Fig. 2c and d). Sediment temperatures follow f l u c t u a t i o n s in air temperature, but with a marked time lag. At a

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depth of 5 cm, maximum and minimum temperatures occur some two hours later than air maxima and minima, while at 25 cm depth the time lag is in the order of four hours. At 25 cm depth, the greatest diurnal fluctuations are seen when sediment temperatures are increasing (Fig. 2a). Oocytes are present in appreciable numbers in the coelomic cavity from the end of March until spawning, which in Dublin Bay occurs epidemically between October and December (HowtE, 1961). Vitellogenic oocytes, of between 50 and 150 #m diameter, were found between the end of April and early November, but accounted for a significant proportion of the oocyte population during June, July and August only (Fig. lb). In female worms, these three months may be assumed to represent the period of maximal nutrient demand. The majority of the worm population therefore undergoes vitellogenesis during the period when the sediment temperatures are rising or maximal (cf. Fig. l a and b). However, there is considerable diversity in the stage of gamete development between worms (Fig. 3). Synchronisation only takes place during September when ambient temperatures have begun to fall. By early October all females are in the postvitellogenic phase. In both 1980 and 1981 spawning started in early November; in 1980 mean sediment temperature at 5 cm depth was 7.4°C, and in 1981 mean temperature was 8.5°C at 5 cm depth and 11.5°C at 25 cm depth during spawning. 3.2. BIOCHEMICAL COMPOSITION Coelomic fluid protein.--In male worms, coelom-

ic fluid protein levels were stable, in the region of 0.57 mg.m1-1, from April to the end of June. From July on, all male worms analysed contained sperm morulae, and protein levels increased steadily to 3.8 m g . m l - 1 in October (Fig. 4a). A further increase gave a peak of 7.8 mg.ml-1 immediately prior to spawning. In contrast with this result, RASHAN (1980) found that coelomic fluid protein levels in male worms dropped prior to spawning, and found a simultaneous decline in the number of protein fractions present. This anomaly could have arisen ~.g-i

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BIOCHEMICAL COMPOSITION IN ARENICOLA

from differences in technique. RASHAN (1980) used Lowry's method of protein determination, while in this study protein was estimated by determination of nitrogen and values include nonprotein nitrogen. After spawning, protein levels fell sharply to a basal level of 0.54 mg.ml-1 In female worms, as in males, coelomic fluid protein levels were stable from April to the end of June. During July, protein levels rose sharply and, with some fluctuation, remained high until spawning. When sufficient worms at different stages of oogenesis were present in the population at the same time, they were analysed separately. Groups of worms containing oocytes of larger diameter showed consistently higher coeIomic fluid protein levels (Fig. 4b). As these results suggested a relationship between coeIomic fluid protein level and the stage of development of gametes, as indicated by oocyte diameter, a further investigation was carried out on a series of individual worms collected in August. The worms were weighed, a sample of 50 oocytes measured for each individual, and coeIomic fluid protein estimated by determination of nitrate. As the relationship between median oocyte diameter and coelomic fluid protein was non-linear, the data were transformed logarithmically (Fig. 5). Partial correlation coefficients were as follows:

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Thus there was a positive, significant relationship between median oocyte diameter and coeIomic fluid protein. Fluctuations in protein level in the pooled samples may be due to variation in weights of the worms sampled; these parameters were positively, although not significantly related (p-0.08). The low protein values seen in September may be due to migration of worms into, and out of the sampling area at this time, changing the weight frequency distribution of the population sampled (MAYES, 1983). Tissue protein.--In both sexes, there is evidence of an inverse relationship between protein levels in the coelomic fluid (above) and in the gut, while protein in the body wall remains relatively stable throughout the annual cycle (Fig. 4a and b). In the females, gut protein levels showed a general decline through summer and autumn, from 609

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mg.g -1 in early June to 410 mg.g -1 immediately post spawning in November (Fig. 4b). A subsequent increase gave a maximum value in January. In males worms, gut protein levels were maximal in May at 654 mg.g -1, and declined, with a subsidiary peak in early November, to the minimal value of 350 mg.g -1 after spawning (Fig. 4a). By January, gut protein had increased to 514 mg.g -1 Lipid.--In the gut tissue, lipid levels rose steadily from April till the end of June, and in both sexes remained fairly stable at about 190 mg.g-1 during July and August (Fig. 6). P,eak values occurred in October, and were followed by a sharp decline. The January values were identical to those of the previous April. Lipid levels in the body wall of both sexes increased during the spring and summer, but changes were less marked than in the gut tissue. In contrast with gut tissue, lipid levels in the body wall tissue of both sexes were higher in January than in the preceding April. Thus, lipid levels, particularly in gut tissue, showed a marked annual cycle. Such large fluctuations suggest a variation in levels of reserve lipids, as structural lipids can be expected to remain relatively constant through time.

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4. DISCUSSION

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Glycogen.--Glycogen levels in the body wall were higher than in the gut, and showed greater fluctuation through the year (Fig. 7). DALES (1958) found that glycogen levels in both body wall and gut tissues fell under anaerobic conditions. DE VooYs (1975) found high levels of glycogen in the gut tissue of juvenile lugworms, and thought this to be due to the greater amount of time spent in anaerobic respiration in juveniles because they occur higher up on the shore than adults and are thus uncovered for longer periods at low tide. If glycogen levels in lugworms are related to their position on the shore, as suggested by DE VOOYS'S results, then fluctuations in glycogen levels seen at Booterstown may be due in part to migration of worms from higher up the shore into the sampling area. Such migrations were noted in late May/early June and in October, at which times glycogen levels peaked (MAYES, 1983).

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The sediment temperature record suggests that sediment acts as a heat sink, absorbing periodic high heat inputs at the surface, so that the average temperature at depth exceeds that at the surface during late spring and summer. It is significant that the summer maximum at 25 cm occurs later than at 5 cm. Peak sediment temperatures occurred in August, some two months later than in The Netherlands (DE WILDE & BERGHUTS, 1979). This discrepancy is probably due to the fact that the two temperature records were made in different years and at different geographic locations. Proliferative activity in the lugworm testis, as measured by mitotic index, is low during the winter and rises during the spring. By the end of April proliferative activity is significant and continues to increase to maximum levels in June (HOWlE & MCCLENAGHAN, 1965; OLIVE, 1972). As a result both male and female gametes appear in increasing numbers in the coelom in late March and early April. In Dublin Bay, sediment temperature begins to rise in early March so that both proliferation and release of gametes into the coeIom occur under conditions of increasing ambient temperature. In the absense of any evidence for endocrine control of gamete proliferation (OLIVE, 1972), it seems possible that the initiation of reproductive activity is the consequence of a general increase in metabolic activity in the spring due to increasing temperature and possibly food supply. CABLE (1976) has shown that feeding activity (as measured by faecal production) is ten times lower in winter than in the summer. He attributes this seasonal difference to a combination of low temperature and food availability during the winter. During the summer, considerable diversity is seen in the stage of gamete development between individual female worms. Synchronisation, which is a prerequisite for epidemic spawning, takes place during September. If gamete growth rates are positively related to temperature, individuals in which gamete development starts relatively late may develop more quickly under conditions of rising temperature. Such a relationship would produce synchronization at the population level during the period of growth (DALY, 1978; OLIVE, 1980). This relationship may be relevant to the lugworm, although synchronization eventually occurs during September when ambient temperature is declining. It

BIOCHEMICAL COMPOSITION IN ARENICOLA

is also possible that the reversal of the direction of temperature change provides an exogenous cue which brings about synchronization. Temperature does not appear to be a direct stimulus for spawning (HOwlE, 1984) but there is some evidence that it may be limiting, in that spawning will not occur if ambient temperatures remain above a certain level. Limiting temperatures of 15°C (FARKE & BERGHUIS, 1979) in the Wadden Sea, and 13°C for the Booterstown population (HOWlE, 1963), have been suggested. In 1980, sediment temperature at 5 cm depth during spawning was 7.4°C on average. In 1981, average temperature during spawning was 8.5°C at 5 cm, and 11.5°C at 25 cm depth thus in both years ambient temperature during spawning was below the proposed limiting temperature of 13oC. In ripe lugworms of both sexes, the gametes comprise some 25% of the total wet weight (DE WILDE & BERGHUIS, 1979). This represents a considerable material investment in reproduction and fluctuations in biochemical composition of somatic tissues are to be expected as gametogenesis progresses. The oocytes and sperm morulae are solitary and the quantity of coelomic cells, although increasing during gamete maturation is small in comparison to the quantity of gametes (DALES, 1957). Thus the coelomic fluid appears to be the major immediate source of nutrient available to the developing gametes. As the gametes develop, coelomic fluid protein levels increase in inverse relation to protein in the gut tissue. RASHAN (1980) has shown by histochemical techniques that proteins are the principal components of the lugworm ooplasm. Deposition of protein commences in the previtelIogenic phase when oocytes are 30-40 #m in diameter, and continues until maturity. The results of the present study suggest that the protein (and non-protein nitrogen) requirements of the developing gametes are supplied by the gut tissue, via the coelomic fluid. Lipid levels in the gut tissue also begin to increase in April and are high throughout the period of gamete growth. Deposition of lipid yolk commences when oocytes are 30-40 #m in diameter, and continues through the vitellogenic phase (RASHAN, 1980). Peak lipid values occur in October, at which stage virtually all females are post-vitellogenic. It is unlikely that further deposition of lipid occurs in the gametes at this time so that this peak may represent an overshoot of supply relative to demand. After spawning lipid

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values in the gut fall sharply. DE VOOYS (1975) has described a drop in glycogen levels in May coincident with the appearance of gametes in the coelom, and attributed the declining glycogen level to the demands of gamete production. At Booterstown, gametes appear in the coelom at an earlier date, and there is no evidence of an associated drop in glycogen levels. On the contrary, glycogen rose in both body wall and gut tissues. Glycogen as such does not occur in lugworm oocytes (RASHAN, 1980), and an understanding of its possible role requires a detailed study of carbohydrate utilization in gametogenesis in this species. It appears, therefore, that protein and lipid are the major reserve materials used in gametogenesis in the lugworm, and these reserves are located in the gut tissue, perhaps in the peritoneal chlorogogenous tissue. The mechanism of transfer of nutrient from the gut to the developing gametes in not known. According to DALES (1957, 1963) lipid is removed from the gut epithelium and peritoneum through the blood, or directly by amoebocytes. The concentration of lipid in the gut is similar to that of the coelomocytes in the lugworm, but the latter are thought to be of little significance in the transfer of nutrients to the gametes. It is possible that, as is the case with protein, lipid is available in suspension in the coelomic fluid. Lipid levels in the body wall tissue rose during the summer, but in contrast with the gut tissue, were higher in January than in the preceding April. This annual pattern of storage and utilization suggests that lipid in the body wall functions as a reserve for the period of reduced food intake during the winter, as is the case in Nereis virens (POCOCK et al., 1971).

5. REFERENCES ALLEN, E.S., 1974. Chemical analysis of ecological materials. Blackwell Scientific Publications. AMERICANPUBLICHEALTHASSOCIATION,1975. Standard methods for the examination of water and wastewater (14th ed.). Amer. Health Assoc., Washington. BLIGH, E.G. & W.J. DYER,1959. A rapid method of total lipid extraction and purification.--Can. J. Biochem. (Physiol.) 37:911-917. CADI~E, G.C., 1976. Sediment reworking by Arenicola marina on tidal flats in the Dutch Wadden Sea.-Neth. J. Sea Res. 10: 440-460. DALES, R.P., 1957. Preliminary observations on the role of the coelomic cells in food storage and trans-

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port in certain polychaetes.--J, mar. biol. Ass. U.K. 36: 91-110. ,1958. Survival of anaerobic periods by two intertidal polychaetes, Arenicola marina (L.) and Owenia fusiformis Delle Chiaje.--J. mar. biol. Ass. U.K. 37: 521-529. - - - - , 1963. Annelids. Hutchison and Co., London: 1-200. DALY, J.M., 1978. The annual cycle and the short term periodicity of breeding in a Northumberland population of Spirorbis spirorbis (Polychaeta: Serpulidae).--J, mar. biol. Ass. U.K. 58" 161-176. FARKE, H. & E.M. BERGHUIS,1979. Spawning, larval development and migration of Arenicola marina under field conditions in the western Wadden Sea.--Neth. J. Sea Res. 13: 529-535. HOWIE, D.I.D., 1961. The spawning of Arenicola marina (L.), I1. Spawning under experimental conditions.--J, mar. biol. Ass. U.K. 41: 127-144. - - - - , 1963. Experimental evidence for the hormonal stimulation of the ripening of the gametes and spawning in the polychaete Arenicola m a r i n a . - Gen. comp. Endocr. 3: 660-668. - - - - , 1966. Further data relating to the maturation hormone and its site of secretion in Arenicola marina (L.).--Gen. comp. Endocr. 6: 347-361. - - - - , 1984. The reproductive biology of the lugworm, Arenicola marina L.--Fortschr. Zool. 29: 247-263. HOWIE, D.I.D. & C. MCCLENAGHAN,1965. Evidence of a feedback mechanism influencing spermatogonial division in the lugworm Arenicola marina (L.).-Gen. comp. Endocr. 5: 40-44. MAYES, E., 1983. Biochemical and environmental aspects of the reproductive cycle in the lugworm Arenicola marina L. M.Sc. Thesis, University of Dublin. MEIJER, L. & M. DURCHON, 1977. Controle neurohormortal de la maturation ovocytaire chez Arenicola marina (Annelide Polychete). l~tude in vitro.--C.r. hebd. Seanc. Acad. Sci., Paris 285: 377-380. OLIVE, P.W.J., 1972. Regulation and kinetics of spermatogonial proliferation in Arenicola marina (Annelida, Polychaeta), I. The annual cycle of mitotic index in the testis.--Cell Tissue Kinet. 5: 245-253.

- - - - , 1980 Environmental control of reproduction in polychaeta: experimental studies of littoral species in north east England. In: W.H. CLARK & T.S. ADAMS. Advances in Invertebrate Reproduction. Elsevier, North Holland: 37-51. OLIVE, P.W.J. & R.B. CLARK, 1978. Physiology of reproduction. In: P.J. MILL. Physiology of annelids. Academic Press, London: 271-368. POCOCK, D.M.-E., J.R. MARSDEN & J.G. HAMILTON, 1971. Lipids in an intertidal polychaete and their relation to the maturation of the worm.--Comp. Biochem. Physiol. 39A: 683-697. POLLACK, H., 1979. Populationsdynamik, Produktivit~it und Energiehaushalt des Wattwurms Arenicola marina (Annelida, Polychaeta).--Helgol&nder wiss. Meeresunters. 32: 313-358. RASHAN, L.J., 1980. Microscopic and biochemical aspects of vitellogenesis in the lugworm Arenicola marina L. Ph.D. Thesis, University of Dublin. RASHAN, L.J. & D.I.D. HOWIE, 1982. Vitellogenesis in the lugworm Arenicola marina L. I. Cytological and ultrastructural observations.--Int. J. Invert. Reprod. 5: 221-231. SCHEINER, D., 1976. Determination of ammonia and Kjeldahl nitrogen by indophenol method.--Water Res. 10: 31-36. SOLORZANO,L. & J.H. SHARP,1980. Determination of total dissolved nitrogen in natural waters.--Limnol. Oceanogr. 25: 751-754. VOOYS, C.G.N. DE, 1975. Glycogen and total lipids in the lugworm (Arenicola m a r i n a ) i n relation to reproduction.--Neth. J. Sea Res. 13: 311-319. WILDE, P.A.W.J. DE & E.M. BERGHUIS, 1979a. Spawning and gamete production in Arenicola marina in the Netherlands, Wadden Sea.--Neth. J. Sea Res. 13: 503-511. - - - - , 1979b. Cyclic temperature fluctuations in a tidal mud-flat. In: E. NAYLOR & R.G. HARTNOLL.. Cyclic phenomena in marine plants and animals. Pergamon Press, Oxford: 435-441. ZWAAN,A. DE & D.I. ZANDEE,1972. Body distribution and seasonal changes in the glycogen content of the common sea mussel Myti/us edu/is.--Comp. Biochem. Physiol. 43A: 53-58.