Assessment of field fertilization success in the infaunal polychaete Arenicola marina (L.)

Summary

Previous studies on the fertilization rates in externally fertilizing marine invertebrates have been concerned principally with free spawning epifaunal organisms (e.g., Babcock et al., 1992). A technique has been developed to investigate fertilization success in the intertidal infaunal polychaete Arenicola marina. The spawning period at the East Sands, St Andrews, is predictable from previous data, and this facilitates the study of spawning behaviour. Female worms spawn within their burrows, where the eggs may remain for several days. Male worms release sperm puddles on to the surface of the sediment at low water. The sperm is carried on the incoming tide over the surface of the sediment where it is actively drawn into female burrows by pumping. The decrease in sperm concentration as the tide comes in has been measured, and it is estimated that there are typically about 10⁶ sperm.ml^?1 in the water column and available to the female at 1 min after covering by the incoming tide. By placing the worms into artificial plastic burrows with removable stoppers, they can be transplanted into the field prior to spawning, and removed afterwards whilst retaining all the contents of the tube (worms, gametes, sediment and water).Preliminary data on the fertilization success suggests that it is highly variable (between 0% and 90%), with typical values of 40–60% for Arenicola marina. Laboratory assays investigating fertilization rate with varying sperm concentration have shown that there is a marked increase in success at 10⁴ and 10⁵ sperm per ml, with values of 30–70%. The field data therefore appear to be consistent with laboratory studies.     

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     

Evidence for a coelomic maturation factor controlling oocyte maturation in the polychaete Arenicola marina (L.)

Summary

Previous studies on Arenicola marina suggested that oocyte maturation was induced by a single maturation hormone from the prostomium. This maturation hormone was thought to act directly on the oocyte (Meijer and Durchon, 1977), A recently described species, Arenicola defodiens (Cadman and Nelson-Smith, 1993), morphologically very similar to A. marina, has been found at the sampling sites described by Meijer and Durchon (1977). Results presented here from studies on British populations of Arenicola marina show that in this species, oocyte maturation is controlled by two hormonal steps. The first step involves the prostomial maturation hormone. The second step depends on a maturation inducing substance in the coelomic fluid. We will refer to this as the coelomic maturation factor (CMF). A reliable in vitro assay for oocyte maturation in the lugworm Arenicola marina has been adopted. It utilizes fluorescence staining of the chromosome material with DNA labelling dyes (Hoechst 33342 and 33258). Maturation of oocytes in A. marina involves germinal vesicle breakdown (GVBD). This is accompanied by the movement of chromosomes from late prophase to metaphase of meiosis I and chromosome condensation. The chromosomes are stained brightly by the dyes and their relative positions can be easily identified so that mature and immature eggs can be distinguished by the differences in chromosome position and form. The development of the in vitro fluorescence assay has enabled us to demonstrate that there are two endocrine steps involved in the induction of oocyte maturation. We have begun the characterization of CMF, and data show this to be a thermolabile molecule with a molecular mass greater than 10 kd.     

The effect of hydrogen peroxide on isolated body wall of the lugworm Arenicola marina (L.) at different extracellular pH levels

The effect of hydrogen peroxide on the rate of tissue oxygen consumption, on intracellular pH (pH(i)) and on malondialdehyde (MDA) accumulation was studied in isolated body wall tissue of the lugworm Arenicola marina (L.). H2O2 effects were investigated at various levels of pH(i) by changing medium pH (pH(e)). The largest decrease of tissue oxygen consumption (by 17% below controls), as well as the highest degree of MDA accumulation (four-fold compared to control values) after H2O2 exposure were found at acidic pH(e) of 6.4. This was attributed to the higher redox potential of H2O2 in acidic solutions. Oxygen consumption at alkaline pH(e) (8.5) was not affected by H2O2. MDA accumulation in the tissue was considerably lower than at pH(e) 7.4 or 6.4. Despite pH dependent alterations of H2O2 redox potential, we observed more or less constant pH(e) independent acidification of the tissue upon exposure to H2O2. We attributed the acidification to an inhibition of ATP consuming proton equivalent ion transport across the cellular membrane. Inactivation of carrier proteins is discussed to be responsible for the decrease in tissue oxygen consumption. However, with a larger effect on oxygen consumption at acidic pH(e) values, the latter may not be the only explanation, but additional impairment of other energy demanding processes may be involved.     

Does seasonal reproduction occur at the optimal time for fertilization in the polychaetes Arenicola marina L. and Nereis virens Sars?

Summary

Many marine invertebrates exhibit highly seasonal and synchronised reproduction, with offspring production often being confined to just two or three days each year. Several models have been proposed to explain the fitness benefits of this reproductive pattern, many of which assume enhanced offspring survival due to temperature constraints placed on fertilization and development at other times of the year. In this investigation the temperature limits and optimum for fertilization were determined for two polychaete species, Arenicola marina and Nereis virens. These two polychaete species are exposed to the same environmental conditions throughout the year, yet breed at very different times. Other seasonal impacts on fertilization, i.e., reduced salinity due to rainfall and the effect of sub-zero temperatures on sperm of A. marina, were also investigated. In both A. marina and N. virens fertilization success was significantly influenced by temperature, with the maximum success recorded at 15–18°C. The ambient seawater temperature at the time of natural spawning for both worms is around 10°C, which means that both species are spawning right at the lower limit for maximum fertilization. Salinity and exposure of A. marina sperm to sub-zero temperatures were also found to influence fertilization success, but only at levels that would not be experienced by these polychaetes under natural conditions at the time of spawning. These results suggest there must be additional selective pressures acting on the fitness of these two polychaetes causing A. marina to breed later than, and N. virens to breed earlier than, the optimum time for fertilization. A. marina apparently waits as late as possible to maximise adult fecundity and survival. N. virens breeds as soon as it can achieve high fertilization to maximise larval and juvenile competitiveness.     

Diclidophora merlangi and Entobdella soleae: egg production and oxygen consumption at different oxygen partial pressures

The rates of respiration and of egg production of the monogenean trematodes Diclidophora merlangi and Entobdella soleae were compared at different oxygen partial pressures. In D. merlangi the respiratory rate declined sharply as the oxygen pressure in the water fell and egg production almost ceased at pO₂s little below air saturation. Diclidophora lives on the gills of a pelagic fish and the likelihood that the parasite normally has air saturated water passing over it is discussed. Both respiratory rate and egg production in E. soleae were almost independent of ambient pO₂. Entobdella lives on the undersurface of a flat fish, which spends part of its time buried in sandy mud.     

Oxygen consumption kinetics of Nitrosomonas europaea and Nitrobacter hamburgensis grown in mixed continuous cultures at different oxygen concentrations

Chemolithotrophic ammonium- and nitrite-oxidizing bacteria are dependent on the presence of oxygen for the production of nitrite and nitrate, respectively. In oxygen-limited environments, they have to compete with each other as well as with other organotrophic bacteria for the available oxygen. The outcome of the competition will be determined by their specific affinities for oxygen as well as by their population sizes. The effect of mixotrophic growth by the nitrite-oxidizing Nitrobacter hamburgensis on the competition for limiting amounts of oxygen was studied in mixed continuous culture experiments with the ammonium-oxidizing Nitrosomonas europaea at different levels of oxygen concentrations.The specific affinity for oxygen of N. europaea was in general higher than of N. hamburgensis. In transient state experiments, when oxic conditions were switched to anoxic, N. hamburgensis was washed out and nitrite accumulated. However, grown at low oxygen concentration, the specific affinity for oxygen of N. hamburgensis increased and became as great as that of N. europaea. Due to its larger population size, the nitrite-oxidizing bacterium became the better competitor for oxygen and ammonium accumulated in the fermentor. It is suggested that continuously oxygen-limited environments present a suitable ecological niche for the nitrite-oxidizing N. hamburgensis.     

In vivo nuclear magnetic resonance studies on the lugworm Arenicola marina. I. Free inorganic phosphate and free adenylmonophosphate concentrations in the body wall and their dependence on hypoxia

The cytoplasmic concentrations of free inorganic phosphate and free AMP in the body wall of the lugworm Arenicola marina were estimated in order to verify their proposed regulatory role in glycogenolysis during anaerobiosis (Kamp 1993). Using in vivo ³¹P nuclear magnetic resonance spectroscopy the concentration of free inorganic phosphate was determined to be 4.7±0.7 mmol·1^-1 (±standard deviation, n=3) varying with season (Juretschke and Kamp 1995). These values were two to three times lower than those measured in perchloric acid extracts. In contrast, values for the phosphagen phosphotaurocyamine assessed biochemically in the extracts and by in vivo nuclear magnetic resonance were very similar. During the transition from normoxia to hypoxia the concentration of free inorganic phosphate increased to the same extent and at the same rate as the concentration of phosphotaurocyamine decreased. A discrepancy was also found for the biochemically determined AMP and ADP concentrations in the extract and those derived from the equilibrium constants of the taurocyamine kinase (ADP_free) and adenylate kinase (AMP_free) reactions. Calculated concentrations of ADP_free and AMP_free in normoxic specimens were about two or even four orders of magnitude lower than the values determined in extracts. During hypoxia the concentrations of AMP and ADP increase moderately when measured biochemically in extracts, while the values for ADP_free and AMP_free rise three- and nine-fold during the first 3 h of hypoxia. Thereafter, the levels stay constant due to a progressive acidosis. If during hypoxia pH_i is stabilized by addition of buffering substances to the incubation medium, both ADP_free and AMP_free rise continuously. The significant changes found for the concentrations of free inorganic phosphate and AMP_free support their assumed regulatory role in glycogenolysis during anaerobiosis, though these AMP_free values seem too low to actually activate glycogen phosphorylase. The strong effect of pH_i on the levels of ADP_free and AMP_free suggest a mechanism by which acidosis prevents a continuing increase of glycogenolysis (ATP-producing pathway) during prolonged anaerobiosis (protective effect of acidosis).Abbreviations ADP _free cytoplasmic adenosine diphosphate - AK adenylate kinase - AMP _free cytoplasmic adenosine monophosphate - CK creatine kinase - GPase glycogen phosphorylase - MW molecular weight - Int P _i integral of P_i-signal - NMR nuclear magnetic resonance - P _i-free cytoplasmic inorganic phosphate - PCA perchlori acid - PFK 6-phosphofructokinase - PME phosphomonoester - PPA phenylphosphonic acid - (P)Tc (phospho)taurocyamine - S _f saturation factor - Sw sea water - TcK taurocyamine kinase - TRA triethanolamine - TRIS tris(hydroxymethyl)aminomethane     

Temperature induced anaerobiosis in two populations of the polychaete worm Arenicola marina (L.)

Temperature dependent changes in the mode of energy metabolism and in acid-base status were studied in the range from −1.7 to 26 °C in two populations of Arenicola marina collected in summer as well as in winter from intertidal flats of the North Sea (boreal) and the White Sea (subpolar). Extreme temperatures led to an accumulation of anaerobic end products, indicating the existence of both a low and a high critical temperature, beyond which anaerobic metabolism becomes involved in energy production. In summer animals from the North Sea the high critical temperature was found at temperatures above 20 °C, and the low critical temperature below 5 °C. Latitudinal or seasonal cold adaptation lead to a more or less parallel shift of both high and low critical temperature values to lower values. Between critical temperatures intracellular pH declined with rising temperature. Slopes varied between −0.012 and −0.022 pH- units/°C. In summer animals from the North Sea, the slope was slightly less than in White Sea animals, but differences appeared independent of the season. However, slopes were no longer linear beyond critical temperatures. A drop in intracellular pH at low temperatures coincided with the accumulation of volatile fatty acids in the body wall tissue of North Sea animals. A failure of active pH_i adjustment is held responsible for the reduced ΔpH_i/ΔT at temperatures above the high critical temperature. Extracellular pH was kept constant over the whole temperature range investigated. The ability of North Sea animals to adapt to temperatures beyond the critical temperature is poor compared to White Sea specimens. The larger range of temperature fluctuations at the White Sea is seen as a reason for the higher adaptational capacity of the subpolar animals. A hypothesis is proposed that among other mechanisms critical temperature values are set by an adjustment of mitochondrial density and thus, aerobic capacity. Accepted: 20 August 1996     

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     

Policy plans and management measures to restore eelgrass (Zostera marina L.) in the Dutch Wadden Sea

The Dutch Wadden Sea has been changed dramatically over the last centuries by human activities like land reclamation and different forms of fishery. This has, amongst other things, led to changes in the number of biological communities. One of the changes was the near extinction of eelgrass (Zostera marina L.) in the Dutch Wadden Sea. The deterioration of the area led to policy plans in the late 1980s that aimed at restoring the original natural communities of which the eelgrass community was one. This paper presents a restoration strategy which contains a selection procedure for suitable transplantation sites. The selection procedure is based on factors such as sediment composition, exposure time, current velocity and wave action. These were combined in a GIS-based map integrating these factors. One important action in the restoration process is to increase the number of freshwater discharge points to meet the requirements of the brackish water community in general and the growing conditions for eelgrass in particular. Received: 27 October 1999 / Received in revised form: 3 February 2000 / Accepted: 3 February 2000     

高等植物大叶藻研究进展及其对海洋沉水生活的适应

综述和讨论了目前对海洋沉水植物大叶藻的研究进展 ,主要包括 :(1 )形态解剖结构特点 ,(2 )基本生理研究 ,(3 )耐盐机理 ,(4)生存限制因子 ,(5 )问题与展望。其中着重讨论了大叶藻与海洋沉水生活相适应的一些特点 ,特别是其对海水盐度的适应机理。     

Impact of acute temperature transition on enzyme activity levels,oxygen consumption,and exogenous fuel utilization in sea raven (Hemitripterus americanus) hearts

Summary The impact of an acute temperature transition between 5 °C and 15 °C on energy metabolism and performance of sea raven (Hemitripterus americanus) heart was assessed. Maximal in vitro activity of hexokinase was 1.2 and 3.7 mol · min^-1 · g^-1 at 5 °C and 15 °C, respectively. Carnitine palmitoyl transferase and carnitine palmitoleoyl transferase activities were 0.07 mol · min^-1 · g^-1 at 15 °C and declined substantially at 5 °C. Oxygen consumption and power output of perfused isolated hearts offered glucose alone as a metabolic fuel decreased significantly between 15 °C and 5 °C. When palmitoleate was included in the perfusion medium, oxygen consumption and power development remained constant between 15 °C and 5 °C, suggesting that glucose alone was not an adequate metabolic fuel at low temperature. However, maximal in vitro activity of HK implied that the catalytic potential at this locus was quite capable of meeting demands of carbon flow, while the maximal in vitro activity of the carnitine acyl CoA transferases implied that fatty acid metabolism should be greatly compromised at low temperatures. In an effort to resolve the contradiction, hearts were perfused with medium containing ¹⁴C-glucose or ¹⁴C-palmitate. Rates of ¹⁴CO₂ production from labelled metabolic fuels could account for only about 2% of the oxygen consumption rates. Most of the label from ¹⁴C-glucose was incorporated into the glycogen and lipid fractions and label from ¹⁴C-palmitate was incorporated into the lipid fraction. The net incorporation rates of label into intracellular pools were temperature insensitive over the range 5–15 °C. The incorporation of exogenous glucose into the lipid fraction suggests that activity of the entire glycolytic pathway was maintained over the temperature range. Thus, the relatively low rate of oxygen consumption of hearts perfused with glucose alone as an exogenous substrate cannot be attributed to a limitation of glucose catabolism. The alternative explanation is that the presence of fatty acids induces an increase in oxygen consumption, especially at 5 °C. It is speculated that this is due to alterations in Ca²⁺ balance.Abbreviations ATPase adenosine triphosphatase - BSA bovine serum albumin - CoA coenzyme A - C palmitoyl T carnitine palmitoyl transferase - CS citrate synthase - HK hexokinase - MO oxygen consumption - PFK phosphofructokinase - PO ₂ oxygen partial pressure     

Life stage variations in insecticidal susceptibility and detoxification capacity of the beet webworm, Pyrausta sticticalis L. (Lep., Pyralidae)

Abstract:  Adults and larvae of the beet webworm Pyrausta sticticalis (Lep., Pyralidae) are susceptible in varying degrees to insecticides of different chemical classes. Comparative toxicity tests as well as the synergistic effects of piperonyl butoxide, S,S,S-tributyl phosphorotrithioate and diethylmaleate showed that variation in toxicities of beet webworm developmental stages to organophosphorus compounds and carbaryl is partly due to the activities of detoxification enzymes. Specific activities of monooxygenases, esterases and glutathione S-transferase were higher in fifth instars than in adults. Studies of the post-treatment fate of ¹⁴C-labelled malathion and ³H- trans -permethrin after application indicated that both compounds had a rapid rate of cuticular penetration, internal accumulation and excretion of applied toxicants and their metabolites by larvae. Adults of beet webworm had a lower sensitivity of acetylcholine esterase to the inhibition of carbaryl than the larvae.     

Effects of temperature on oxygen consumption and heart rate in the semi‐terrestrial crab,Ocypode sa ratan (Forskål)

The effect of temperature on the rate of oxygen consumption and heart rate has been investigated in the Red Sea, semi‐terrestrial crab, Ocypode saratan. The Q₁₀ values for both oxygen consumption and heart rate were often <2 and were relatively constant over a wide range of temperature. This suggests that O. saratan is well adapted for life in a high temperature environment. It was noted, however, that behavioural mechanisms were important in reducing the degree of temperature stress that could be experienced in the field. The rates of oxygen consumption of immersed crabs were significantly lower than those of crabs maintained in air at the same temperature.     

Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia

Anaerobic metabolism in the limnic annelid Hirudo medicinalis L. was investigated by direct and indirect calorimetry. During long-term severe hypoxia, the rate of heat dissipation was reduced up to 13% of the aerobic rate. At the same time, the rate of ATP turnover was reduced to about 30% of the aerobic rate, indicating that metabolic depression is an important mechanism to ensure survival of the leech during environmental anaerobiosis. Heat dissipation during hypoxia was monitored under two experimental conditions, favouring either concomitant hypocapnia (continuous N₂ bubbling) or hypercapnia (self-induced hypoxia). The reduction in heat dissipation during hypocapnic hypoxia was less pronounced than during hypercapnic hypoxia, indicating that the different experimental conditions may influence anaerobic metabolism and the extent of metabolic depression. Biochemical analysis of known anaerobic substrates and endproducts provided the basis for indirect calorimetry during self-induced hypoxia. From changes in metabolites, the expected heat dissipation was calculated for initial (0–8 h) and long-term severe hypoxia (8–72 h). During the initial period, the calculated heat dissipation fully accounted for direct calorimetric determination. During long-term hypoxia, only 71% of the measured heat production could be explained from biochemical analysis of metabolites. Therefore, an additional unknown endproduct cannot be excluded, especially when anaerobic ammonia production and analysis of the carbohydrate balance are considered.Abbreviations APW artificial pond water - HPLC high-performance liquid chromatography - fw fresh weight - HP heat production - HD heat dissipation - MR metabolic rate     

Stimulation of ethylene production and gas-space (aerenchyma) formation in adventitious roots of Zea mays L. by small partial pressures of oxygen

Adventitious roots of two to four-weekold intact plants of Zea mays L. (cv. LG11) were shorter but less dense after extending into stagnant, non-aerated nutrient solution than into solution continuously aerated with air. Dissolved oxygen in the non-aerated solutions decreased from 21 kPa to 3–9 kPa within 24 h. When oxygen partial pressures similar to those found in non-aerated solutions (3, 5 and 12 kPa) were applied for 7 d to root systems growing in vigorously bubbled solutions, the volume of gas-space in the cortex (aerenchyma) was increased several fold. This stimulation of aerenchyma was associated with faster ethylene production by 45-mm-long apical root segments. When ethylene production by roots exposed to 5 kPa oxygen was inhibited by aminoethoxyvinylglycine (AVG) dissolved in the nutrient solution, aerenchyma formation was also retarded. The effect of AVG was reversible by concomitant applications of 1-aminocyclopropane-1-carboxylic acid, an immediate precursor of ethylene. Addition of silver nitrate, an inhibitor of ethylene action, to the nutrient solution also prevented the development of aerenchyma in roots given 5 kPa oxygen. Treating roots with only 1 kPa oxygen stimulated ethylene production but failed to promote gas-space formation. These severely oxygen-deficient roots seemed insensitive to the ethylene produced since a supplement of exogeneous ethylene that promoted aerenchyma development in nutrient solution aerated with air (21 kPa oxygen) failed to do so in nutrient solution supplied with 1 kPa oxygen. Both ethylene production and aerenchyma formation were almost completely halted when roots were exposed to nutrient solutions devoid of oxygen. Thus both processes require oxygen and are stimulated by oxygen-deficient surroundings in the 3-to 12-kPa range of oxygen partial pressures when compared with rates observed in air (21 kPa oxygen).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine