Contrasting breeding periodicity of nearby populations of the lugworm,Arenicola marina (Annelida,Polychaeta)

Summary

The breeding cycles of lugworm taken from three locations on the east coast of Ireland have been investigated through examination of coelomic fluid samples which contain the gametes, taken at monthly intervals. These reveal major differences in the timing and duration of breeding between the populations despite their geographic propinquity. The differences in breeding cycle cannot be ascribed to varietal or species differences and may arise from the different habitats occupied by the lugworm populations studied.     

Mechanisms of sulphide tolerance in the peanut worm,Sipunculus nudus (Sipunculidae) and in the lugworm,Arenicola marina (Polychaeta)

Summary The peanut worm Sipunculus nudus and the lugworm Arenicola marina are inhabitants of intertidal flats. Both species may be exposed to H₂S within their habitat. Sulphide concentrations in the vicinity of A. marina burrows are as high as 340 mol · 1^-1, whereas the pore water in sipuncle areas contains much lower sulphide levels of 13 mol · 1^-1 at most. During in vivo sulphide incubations, H₂S increases within the coelomic fluid of both species. In S. nudus the concentration of total sulphide after 8 h is about 40% of that of the incubation medium containing 200 and 1000 mol · 1^-1, respectively, which is partly due to the acidification of the coelomic fluid by 0.2 pH units during anaerobiosis. After 8 h, the sulphide concentration in A. marina was only 15% of that in the incubation medium containing 1000 mol · 1^-1. When oxygen is available, both species oxidize sulphide to thiosulphate, but in A. marina this capability is more pronounced than in S. nudus. If sulphide is not completely oxidized internally both intertidal worms switch to an anaerobic metabolism as indicated by the accumulation of opines and succinate.Abbreviations ATP adenosine triphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - HPLC high-performance liquid chromatography - wwt wet weight     

Bioelectrical activity in the heart of the lugworm Arenicola marina

Standard microelectrode technique was used to study electrical activity of the isolated heart of the polychaete annelid, Arenicola marina. Typical pacemaker activity with slow diastolic depolarization was observed in all recordings. The average maximum diastolic potential (−58.4 ± 3.2 mV), the average amplitude of the action potential (28.7 ± 4.7 mV) and the average total duration of the action potential (2,434 ± 430 ms) were determined. There has been no gradient of automaticity observed in our studies, which suggests that all regions of the Arenicola heart could possess pacemaker functions. Acetylcholine (ACh) produced a concentration dependent (5 × 10⁻⁸–5 × 10⁻⁵ M) increase of the beating rate via increase in the rate of the diastolic depolarization. ACh (5 × 10⁻⁵ M) increased beating rate by 2.5-fold compared to the control rate. A stronger action of ACh resulted in depolarization, block of action potential generation and contracture of the heart. The non-hydrolysable ACh analog carbacholine (10⁻⁸–10⁻⁶ M) produced similar effects. All effects of ACh and carbacholine were abolished by 5 × 10⁻⁶ M atropine. d-Tubocurarine (5 × 10⁻⁵ M) did not significantly alter effects of ACh or carbacholine. Epinephrine (10⁻⁸–10⁻⁶ M) caused the slowing of pacemaker activity and marked decrease of action potential duration. 10⁻⁶ M epinephrine produced complete cardiac arrest. The effects of epinephrine were not significantly altered by the β-blocker propranolol (5 × 10⁻⁶ M). The β-agonist isoproterenol (10⁻⁷–10⁻⁵ M) and the α-agonist xylometazoline (10⁻⁶–10⁻⁵ M) did not produce significant effects. Thus, cholinergic effects in the Arenicola heart are likely to be mediated via muscarinic receptors, while the nature of adrenergic effects needs further investigation.     

Ultrastructural studies of the hearts in Arenicola marina L. (Annelida: Polychaeta)

Summary The two hearts in Arenicola are capable of great dilation and contraction. The heart wall consists of myoepithelial cells resting on a basal lamina. On the luminal side of the basal lamina is a layer of collagen fibrils. No true endothelium was observed but occasional haemocytes were observed, subjacent to the collagenous layer. A few chloragogen cells are also found peripherally.The myofibrils are of a non-striated type consisting of thick and thin filaments and scattered Z-bodies. The sarcoplasmic reticulum forms a three-dimensional network. Only peripheral couplings were observed. The myofibrils are in contact with the sarcolemma on the luminal side of the cells, constituting a kind of hemidesmosome. The myoepithelial heart muscle is compared with other muscle types described in invertebrates. Supercontraction is discussed.     

Sulfide : quinone oxidoreductase (SQR) from the lugworm Arenicola marina shows cyanide- and thioredoxin-dependent activity

The lugworm Arenicola marina inhabits marine sediments in which sulfide concentrations can reach up to 2 mM. Although sulfide is a potent toxin for humans and most animals, because it inhibits mitochondrial cytochrome c oxidase at micromolar concentrations, A. marina can use electrons from sulfide for mitochondrial ATP production. In bacteria, electron transfer from sulfide to quinone is catalyzed by the membrane-bound flavoprotein sulfide : quinone oxidoreductase (SQR). A cDNA from A. marina was isolated and expressed in Saccharomyces cerevisiae, which lacks endogenous SQR. The heterologous enzyme was active in mitochondrial membranes. After affinity purification, Arenicola SQR isolated from yeast mitochondria reduced decyl-ubiquinone (K(m) = 6.4 microm) after the addition of sulfide (K(m) = 23 microm) only in the presence of cyanide (K(m) = 2.6 mM). The end product of the reaction was thiocyanate. When cyanide was substituted by Escherichia coli thioredoxin and sulfite, SQR exhibited one-tenth of the cyanide-dependent activity. Six amino acids known to be essential for bacterial SQR were exchanged by site-directed mutagenesis. None of the mutant enzymes was active after expression in yeast, implicating these amino acids in the catalytic mechanism of the eukaryotic enzyme.     

Some Ecological Features of the Lugworm Arenicola marina L. (Annelida, Polychaeta) and Its Morphological and Biochemical Adaptations to Burrowing

Hydrochemical conditions developing in the burrows of lugworms (Arenicola marina L.) by the end of low-tide period are analyzed. The behavior of lugworms in the course of gradual decrease in the content of dissolved oxygen is described. Quantitative data on the hemoglobin contents in the blood of lugworms from different regions of the White Sea are presented. Morphological and biochemical adaptations facilitating the survival of these animals in their narrow burrows upon sharp fluctuations of environmental conditions, which are characteristic of the littoral zone, are discussed.     

Blood oxygen transport and metabolism of the confined lugworm Arenicola marina (L.).

Oxygen consumption (MO2), haemoglobin oxygen saturation level (SVO2) and pH (pHv) in prebranchial blood were measured in lugworms experimentally confined in sea water at 15 degrees C. Total blood flow through the gills (Vb) was estimated. For sea water oxygen partial pressure (PwO2) between 120 and 150 Torr MO2, SVO2 and Vb were high and nearly constant. For PwO2 less than 120 Torr, Vb fell quickly, MO2 progressively dropped, and metabolism remained aerobic at the expense of the prebrancial blood oxygen store. For PwO2 less than 50 Torr, Vb and SvO2 values were extremely low, and the low pHv and the modified buffer power of the surrounding sea water showed that anaerobic metabolism was occurring. Changes in respiratory gas exchanges and metabolism during the tidal cycle are deduced from the comparison of these results with data obtained in the field.     

Density-dependent recruitment after winter disturbance on tidal flats by the lugworm Arenicola marina

The polychaete Arenicola marina is abundant and widespread on intertidal sand flats in K?nigshafen (island of Sylt, North Sea). Juveniles overwinter in subtidal channels and then colonize the upper tidal zone above the range of the adults. In the summers of 1995 and 1997, after a mild and a moderate winter, a distinct nursery belt fringing the shoreline was apparent. The severe winter of 1995/96 changed this pattern. Population size of the adults was halved, and in summer 1996 the juveniles were no longer restricted to an upper intertidal belt and settled over a wide range of tidal flats where adults were diminished. This spatial expansion of recruits is assumed to be a density-dependent response. An overcompensation by one-third of the previous population size did not carry over into the next year. Compared to some other species of the tidal-flat fauna, the disturbance effect by the severe winter on the lugworm population was small and brief. Received in revised form: 7 May 2001 Electronic Publication     

In vivo 13C-NMR studies on the metabolism of the lugworm Arenicola marina

13C-NMR natural-abundance spectra of specimens of Arenicola marina obtained, showed seasonal changes in the concentration of some metabolites, with the osmolite alanine as well as triacylglyceride storage compounds present at high concentrations. Glycogen was sometimes only barely detectable due to the low natural abundance level of 13C. Glycogenic metabolism of the lugworm A. marina was studied in vivo by 13C-NMR spectroscopy using 13C-labelled glucose. During recovery from a hypoxic period [1-13C]glucose was incorporated into glycogen. [1-13C]Glucose was injected 5 h after the end of hypoxia to guarantee sufficient and reliable 13C labelling of glycogen. An earlier injection of [1-13C]glucose led to considerably diminished incorporation of 13C-labelled glucosyl units into glycogen, probably due to the consumption of the available glucose as fuel for ATP production. No scrambling of 13C into the C6 position of glycogen was observed, indicating a lack of gluconeogenic activity. 13C was also incorporated into the C3 positions of alanine and alanopine. To assign correctly this last 13C-NMR resonance, the compound was synthesized biochemically. No labelling of glycogen was observed when [3-13C]alanine was injected into the coelomic cavity with similar incubation conditions being used. The 13C of [1-13C]glucose, incorporated into glycogen, showed a very low turnover rate in normoxic lugworms as shown by two 13C(1H)-NMR spectra, one obtained 48 h after the other. On the other hand, in hypoxia lugworms the signal due to 13C-labelled glycogen decreased very rapidly proving a high turnover rate. The disappearance of 13C from glycogen during the first 24 h of hypoxia indicates that the last glycosyl units to be synthesized are the first to be utilized. Lugworms were quite sensitive to the 1H-decoupling field used for obtaining the 13C(1H)-NMR spectra, especially at 11.7 T. Using bi-level composite-pulse decoupling and long relaxation delays, no tissue damage or stress-dependent phosphagen mobilization, as judged by 31P-NMR spectroscopy, was observed.     

Isolation and characterization of five serine proteases with trypsin-, chymotrypsin- and elastase-like characteristics from the gut of the lugworm Arenicola marina (L.) (Polychaeta)

Summary Five proteases were isolated from the digestive fluid of the lugworm, Arenicola marina L. The enzymes (molecular weight 24.0–24.6 kDa) were classified as serine proteases. Three enzymes showed a cleavage specificity corresponding to mammalian trypsin (E.C. 3.4.21.4). One protease possessed a chymotrypsin-like cleavage pattern (E.C. 3.4.21.1), and the fifth preferred cleavage behind short-chain amino acids like an elastase (E.C. 3.4.21.36). Detailed investigations revealed differences in molecular characteristics and cleavage patterns compared to mammalian proteases, especially in the chymotrypsin- and the elastase-like enzymes.Abbreviations APNE N-acetyl-d/l-Phe -naphthyl ester - BANA N-benzoyl-d/l-Arg -naphthylamide - BAPNA N-benzoyl-d/l-Arg-4-nitroanilide - BIGGANA N-benzoyl-l-Ile-l-Glu-Gly-l-Arg-4-nitroanilide - BLPNA N-benzoyl-d/l-Lys-4-nitroanilide - BTEE N-benzoyl-l-Tyr ethyl ester - enzyme T1/T2/T3 trypsin-like enzyme - enzyme ChT chymotrypsin-like enzyme - enzyme E elastase-like enzyme - GPANA N-glutaryl-l-Phe-4-nitroanilide - MUF 4-methylumbelliferryl - MW molecular weight - PMSF phenylmethylsulphonyl fluoride - SAAPPNA N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-4-nitroanilide - SBTI soybean trypsin inhibitor - SPPNA N-succinyl-l-Phe-4-nitroanilide - TAME N-tosyl-l-Arg methyl ester - TFA trifluoracetic acid - TLCK N-tosyl-l-Lys chloromethyl ketone - TPCK N-tosyl-l-Phe chloromethyl ketone - TRIS tris(hydroxymethyl)aminomethane     

Mitochondrial function in seasonal acclimatization versus latitudinal adaptation to cold in the lugworm Arenicola marina (L.)

Previous studies in marine ectotherms from a latitudinal cline have led to the hypothesis that eurythermal adaptation to low mean annual temperatures is energetically costly. To obtain more information on the trade-offs and with that the constraints of thermal adaptation, mitochondrial functions were studied in subpolar lugworms (Arenicola marina L.) adapted to summer cold at the White Sea and were compared with those in boreal specimens from the North Sea, either acclimatized to summer temperatures or to winter cold. During summer, a comparison of mitochondria from subpolar and boreal worms revealed higher succinate oxidation rates and reduced Arrhenius activation energies (Ea) in state 3 respiration at low temperatures, as well as higher proton leakage rates in subpolar lugworms. These differences reflect a higher aerobic capacity in subpolar worms, which is required to maintain motor activity at low but variable environmental temperatures--however, at the expense of an elevated metabolic rate. The lower activity of citrate synthase (CS) found in subpolar worms may indicate a shift in metabolic control within mitochondria. In contrast, acclimatization of boreal lugworms to winter conditions elicited elevated mitochondrial CS activities in parallel with enhanced mitochondrial respiration rates. With falling acclimation temperatures, the significant Arrhenius break temperature in state 3 respiration (11 degrees C) became insignificant (5 degrees C) or even disappeared (0 degrees C) at lower levels of Arrhenius activation energies in the cold, similar to a phenomenon known from hibernating vertebrates. The efficiency of aerobic energy production in winter mitochondria rose as proton leakage in relation to state 3 decreased with cold acclimation, indicated by higher respiratory control ratio values and increased adenosine diphosphate/oxygen (ADP/O) ratios. These transitions indicate reduced metabolic flexibility, possibly paralleled by a loss in aerobic scope and metabolic depression during winter cold. Accordingly, these patterns contrast those found in summer-active, cold-adapted eurytherms at high latitudes.     

CDK1/cyclin B regulation during oocyte maturation in two closely related lugworm species, Arenicola marina and Arenicola defodiens

The molecular mechanisms underlying oocyte maturation in the annelid polychaetes Arenicola marina and Arenicola defodiens were investigated. In both species, a hitherto unidentified hormone triggers synchronous and rapid transition from prophase to metaphase, a maturation process which can be easily reproduced in vitro. Activation of a roscovitine- and olomoucine-sensitive M-phase-specific histone, H1 kinase, occurs during oocyte maturation. Using affinity chromatography on immobilized p9CKShs1, we purified CDK1 and cyclin B from oocyte extracts prepared from both phases and both species. In prophase, CDK1 is present both as an inactive, but Thr161-phosphorylated monomer, and as an inactive (Tyr15-phosphorylated) heterodimer with cyclin B. Prophase to metaphase transition is associated with complete tyrosine dephosphorylation of the cyclin B-associated CDK1, with phosphorylation of cyclin B, and with dramatic activation of the kinase activity of the CDK1/cyclin B complex. We propose that Arenicola oocytes may provide an ideal model system to investigate the acquisition of the ability of oocytes to be fertilized that occurs as oocyte shift from prophase to metaphase, an important physiological event, probably regulated by active CDK1/cyclin B.     

Characterization and peptidase specificity of lugworm (Arenicola cristata) protease C

1. Lugworm protease C further purified by benzamidine-affinity chromatography, exhibited peptidase specificity for arginyl and lysyl bonds. 2. Protease C consisted of a single polypeptide with a mol. wt of ca 23,000 as determined by SDS polyacrylamide gel electrophoresis, exhibited a u.v. absorption maximum at 280 with an (mg/ml) extinction coefficient of 0.93 and fluorescence spectra typical of a protein containing tryptophan, and had an amino acid composition similar to trypsins. 3. The Kms of the cleavages of the arginyl bond of oxidized insulin B chain and of the lysyl bond of the gly23-ala30 fragment were determined to be 0.72 and 0.96 mM; the corresponding kcats were 38 sec-1 and 1.5 sec-1. The Km and kcat for TAME were 0.042 mM, and 110 sec-1. 4. Lugworm protease C was confirmed to be a trypsin.     

In vivo nuclear magnetic resonance studies on the lugworm Arenicola marina. II Seasonal changes of metabolism

In vivo ³¹P- and ¹³C-NMR spectra of the lugworm Arenicola marina (Polychaeta, Annelida) gathered between 1988 and 1994 at different times of the year were evaluated for seasonally dependent metabolic changes. Beside the typical ³¹P-NMR signals of ATP and (phospho)taurocyamine, other seasonally dependent signals were observed: from January to March an intensive signal at 1.4–1.8 ppm was identified as inorganic phosphate compartmented in an acidic intestinal lumen. Between April and September signals at 1.2–1.4 ppm were assigned to phosphodiester. Starting in July males showed a second phosphagen signal [(phospho)creatine of spermatozoa, cf. Kamp and Juretschke (1989a)] whose intensity increased until spawning in September. The (phospho)taurocyamine/ATP ratio was also dependent on the season. In January or February the ratio reached 11, while in summer and autumn the ratio was between 4 and 5. As verified by biochemical assays this effect was caused by a significant decrease of ATP in the lugworm body wall during winter (December–February). The phosphagen (phospho)taurocyamine and the respective unphosphorylated guanidine taurocyamine remained constant throughout the year. Levels of free inorganic phosphate incurred similar changes to ATP. ¹³C-NMR spectra of lugworms showed a dramatic change in lipid stores. They were below the detection limit between January and March but developed into the most intensive signals during summer. The most abundant amino acids, glycine and alanine, were observed throughout the year while glycogen could not be detected in the ¹³C-NMR spectra. After treating tissue extracts with amyloglucosidase, the signals of the hydrolytic product glucose were recorded indicative of NMR-invisible glycogen stores.Abbreviations AN adenine nucleotides - NMR nuclear magnetic resonance - P_i inorganic phosphate - (P)Cr (phospho)creatine - PDE phosphodiester - PME phosphomonoester - PPA phenylphosphonic acid - (P)Tc (phospho)taurocyamine