Metabolite concentrations in electrically stimulated lugworms Arenicola marina

• 1.1. The concentrations of lactate, succinate, alanine, aspartate, acetate and propionate in the lugworm Arenicola marina were measured after 1, 2.5 and 12 hr of continuous electrical stimulation under aerobic and anaerobic conditions.
• 2.2. A continuous increase of the concentrations of alanine and acetate, and a decrease of aspartate occurred during the first 2.5 hr of electrical stimulation. A marked rise of succinate and propionate was observed only in experiments lasting longer than 2.5 hr.
• 3.3. No changes were detected in the concentrations of lactate. Under anoxia the metabolites accumulated at significantly higher rates than under aerobic conditions.

     

Arenicola marina extracellular hemoglobin: a new promising blood substitute

The need to develop a blood substitute is now urgent because of the increasing concern over Europe's BSE outbreak and the worldwide HIV/AIDS epidemic, which have cut blood supplies. Extracellular soluble hemoglobin has long been studied for its possible use as a safe and effective alternative to blood transfusion, but this has met with little success. Clinical trials have revealed undesirable side effects-oxidative damage and vasoconstriction-that hamper the application of cell-free hemoglobin as a blood substitute. We have addressed these problems and have found a new promising extracellular blood substitute: the natural giant extracellular polymeric hemoglobin of the polychaete annelid Arenicola marina. Here we show that it is less likely to cause immunogenic response; its functional and structural properties should prevent the side effects often associated with the administration of extracellular hemoglobin. Moreover, its intrinsic properties are of interest for other therapeutic applications often associated with hemorrhagic shock (ischemia reperfusion, treatment of septic shock and for organ preservation prior to transplantation). Moreover, using natural hemoglobin is particularly useful since recombinant DNA techniques could be used to express the protein in large quantities.     

Sulfide Tolerance and Detoxification in Arenicola marina and Sipunculus nudus

SYNOPSIS. The lugworm Arenicola marina and the peanut worm Sipunculusnudus both inhabit intertidal flats. Sulfide concentrationsin the pore water of the sediment can be as high as 340 µMin the habitat of A. marina. Sulfide levels in the burrow waterand in the coelomic fluid increase during low tide. In contrast,S. nudus is only rarely exposed to sulfide. Neither A. marinanor S. nudus is able to exclude sulfide completely from itsbody. However, both worms accumulate only 15 to 40% respectivelyof the external sulfide concentration during hypoxic 1 mM sulfideincubations. During normoxia, sulfide accumulation is more thanten-fold lower than under hypoxic conditions in both species.When oxygen is available, sulfide is oxidized to thiosulfate.The oxidation rate is about 5 times higher in A. marina thanin S. nudus. A major component of sulfide oxidation is due tothe mitochondria of both species. Mitochondrial sulfide oxidationin the tissues of A. marina is insensitive to low cyanide andto high sulfide concentrations, but can be inhibited by salicylhydroxamicacid. The existence of an alternative terminal oxidase is suggested.When the sulfide level in the body of A. marina and S. nudusincreases above a toxic level, both species survive by switchingto an anaerobic metabolism.     

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.     

Burrowing behaviour in the European lugworm Arenicola marina (Polychaeta: Arenicolidae)

A detailed analysis is presented of the mechanism of burrowing in Arenicola marina (L.). The following were recorded electronically, using further developments of the techniques introduced by Trueman: movement into the sand, internal pressure at various points in the body, external pressure in the surrounding sand. The results are interpreted in relation to direct visual observation of the burrowing process. The structural and functional specializations of the lugworm for burrowing and the factors concerned in the release and control of burrowing activity, are discussed.     

Volatile fatty acid excretion during anaerobiosis in the lugworm Arenicola marina

• 1.1. Accumulation and excretion of propionate and acetate during experimental anaerobiosis were investigated in the lugworm Arenicola marina.
• 2.2. The rate of accumulation and the ratio propionate/acetate were found to be tissue-specific.
• 3.3. The excretion of the volatile fatty acids showed a characteristic time course.
• 4.4. The results of experiments analyzing the role of different organs indicate that the excretion of these metabolites proceeded via the undifferentiated surface of the body.
• 5.5. The rate of excretion depended on the concentration gradient between animal and the ambient water, the chain-length of the fatty acid and the pH of the water. Propionate excretion was inhibited by butyrate.

     

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.     

Comparison of the two different phosphagen systems in the lugworm Arenicola marina

The different phosphagen systems in the lugworm Arenicola marina, the phosphotaurocyamine/taurocyamine kinase system of the body wall and the phosphocreatine/creatine kinase system of the spermatozoa, have been investigated to answer the question whether the change reflects different functional modes of these phosphagen systems. Enzyme analyses have shown that in contrast to the body wall taurocyamine kinase, creatine kinase of spermatozoa exists in at least two different forms which are compartmented in the mitochondria (creatine kinase I) and in the flagellum (creatine kinase II). Creatine kinase I is strongly attached to cell structures which require detergents and high phosphate concentrations for solubilization. The affinities of taurocyamine kinase and creatine kinase for all substrates are very similar except the extremely high K _m for creatine of both creatine kinase I and II. The level of creatine in spermatozoa is fivefold higher than taurocyamine in the body wall at similar phosphorylation potential (ATP/ADO_free) and ATP-buffer capacity (phosphagen/ATP), reflecting the higher equilibrium constants of the creatine kinase reaction compared to that of the taurocyamine kinase reaction (Ellington 1989). The high creatine concentration gives the phosphocreatine/creatine kinase system an advantage over the phosphotaurocyamine/taurocyamine kinase system for transport of energyrich phosphate at high phosphorylation potential by increasing the radial diffusion flux. The maximum diffusive flux of free ADP in spermatozoa is three orders of magnitude below the respiratory ATP production while the creatine flux would allow an unlimited energy transport over the long diffusion distance. In lugworm body wall, however, the low ATP turnover and the low diffusion distances between mitochondria and myosin-ATPases do not require a phosphagen shuttle.Abbreviations ADP _free cytoplasmic adenosine diphosphate - Ap ₅ A P₁, P₅-di(adenosine-5-) pentaphosphate - AK arginine kinase - CK creatine kinase (EC 2.7.3.2) - DTT dithiothreitol - GAPDH glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) - HOADH 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) - IEP isoelectric point - MIM mitochondria isolating medium - P _i-free cytoplasmic inorganic phosphate - (P)Arg (phospho)arginine - (P)Cr (phospho)creatine - (P)Tc (phospho)taurocyamine - SEM scanning electron microscopy - TK taurocyamine kinase - TEM transmission electron microscopy     

Aggression and courtship in Drosophila: pheromonal communication and sex recognition

Upon encountering a conspecific in the wild, males have to rapidly detect, integrate and process the most relevant signals to evoke an appropriate behavioral response. Courtship and aggression are the most important social behaviors in nature for procreation and survival: for males, making the right choice between the two depends on the ability to identify the sex of the other individual. In flies as in most species, males court females and attack other males. Although many sensory modalities are involved in sex recognition, chemosensory communication mediated by specific molecules that serve as pheromones plays a key role in helping males distinguish between courtship and aggression targets. The chemosensory signals used by flies include volatile and non-volatile compounds, detected by the olfactory and gustatory systems. Recently, several putative olfactory and gustatory receptors have been identified that play key roles in sex recognition, allowing investigators to begin to map the neuronal circuits that convey this sensory information to higher processing centers in the brain. Here, we describe how Drosophila melanogaster males use taste and smell to make correct behavioral choices.     

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.     

The Kinetics of Bacteriolysis in the Gut of the Deposit Feeder Arenicola marina

A turbidimetric technique was used to measure the bacteriolytic activities of digestive fluids collected seasonally from the deposit-feeding polychaete Arenicola marina. Lysis of cultured sedimentary bacteria was monitored spectrophotometrically. The kinetics of the lytic reaction were characterized with respect to temperature, pH, reactant concentration, and time. Bacteriolysis generally followed saturation kinetics behavior and was apparently a first-order process with respect to the substrate (bacteria). Deviation from a simple Michaelis-Menten relationship, however, was observed at high substrate levels, at which inhibition of lysis was evident. The lytic rate decreased rapidly and sharply through time as the substrate was depleted. Lytic factors in the gut fluid acted as catalysts; rates increased with concentration of gut fluid, but no depletion in lytic capacity through time was observed. The lytic rate increased exponentially for all seasonal samples over the range of environmental temperatures (4 to 31(deg)C) but showed little relationship with pH under conditions likely to be experienced in situ. The lytic rate also varied among bacterial strains and with culture age.     

Determination by HPLC fluorescence analysis of the natural enantiomers of sex pheromones in the New World screwworm fly, Cochliomyia hominivorax

Abstract.  Bioassays of six racemic synthesized candidate sex pheromone compounds against male New World screwworm Cochliomyia hominivorax (Coquerel) flies showed that the most potent bioactivity was found with 6-acetoxy-19-methylnonacosane and 7-acetoxy-15-methylnonacosane compared with four other isomeric acetoxy nonacosanes and a larger aliphatic ketone. As all these methyl-branched compounds have two asymmetric carbons and four possible enantiomers, characterization of the natural enantiomers was essential. All four enantiomers for the two most bioactive isomers of the natural sex pheromone were synthesized for bioassay. Hydrolysis and derivatization of these enantiomers with different fluorescent reagents was followed by column-switched high-performance liquid chromatography. The use of two linked, reversed-phase columns of different polarity held at sub-ambient temperatures allowed good separation of each enantiomer. This analysis applied to natural material was successful, as (6 R ,19 R )-6-acetoxy-19-methylnonanocosane, and (7 R ,15 R )- and (7 R ,15 S )-7-acetoxy-15-methylnonanocosane were detected in extracts of recently colonized female flies.     

Spawning behavior of Arctic charr (Salvelinus alpinus): Spawning synchrony,vibrational communication,and mate guarding

A mismatch in synchrony between male and female gamete release in external fertilizers can result in reduced or failed fertilization, sperm competition, and reduced paternity. In Arctic charr (Salvelinus alpinus), males can adopt either a guard or sneak tactic resulting in both pre‐ and postcopulatory competition between males with alternative reproduction tactics. Here, spawning behavior of free‐living Arctic charr was video‐recorded, and their reproductive behavior was analyzed. From evaluating 157 spawning events, we observed that females mainly spawned with a guarding male and that the female and the guarding male synchronized timing of gamete release under sperm competition. Although sneakers spawned with higher synchrony than the guarding male in single‐male spawning events, the average sneaker released his milt less synchronized with the female than the guarding male under sperm competition. Approximately 50% of the recorded spawning events occurred under sperm competition, where each event included an average of 2.7 males. Additionally, sneakers were more exposed to sperm competition than guarding males. An influx of males, in close proximity to the female, occurred during the behavioral sequences leading up to egg release, but this influx seemed not dependent on egg release, suggesting that something else than gonadal product attracts sneaker males to the spawning female. Just before and during the actual release of gametes, the spawning couple vibrates their bodies in close contact and it seems likely that this vibrational communication between the spawning couple, which results in a larger amplitude sound wave than seen under regular courting, reveals time of gamete release to sneaker males. Thus, vibrational communication may enable synchrony between the guarding male and the female, and this might be traded against the cost of higher detectability from surrounding sneaker males, eavesdropping in close proximity.     

The pheromonal gland of Lymantria dispar: Morphology and evidence for its innervation

The morphological features of the glandular epithelium that secretes pheromone in the polyphagous pest gypsy moth Lymantria dispar are described by light and electron microscopy. The monolayered gland cells are covered by the folded cuticle of the intersegmental membrane between the 8th and 9th abdominal segments showing neither sites of discontinuity nor distinct openings on its external surface. The cells bear a large, often irregularly shaped nucleus, and contain granules of variable amount and electron‐density. These granules are mostly located in the basal compartment of the cytoplasm, in a labyrinthine zone laying on a basement membrane. The apical membrane of the gland cells bear microvilli and cell–cell contact is established by different junctional structures. Nerve fibers enwrapped in glia are found beneath the basement membrane, in close contact with the secretory cells. This latter finding represents the first evidence of the innervation of the pheromonal gland in L. dispar. J. Morphol. 2009. © 2008 Wiley‐Liss, Inc.     

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.     

Anaerobic metabolism in the lugworm Arenicola marina during low tide: The influence of developing reproductive cells

• 1.1. The extent of anaerobic energy production of Arenicola marina during low tide is dependent on the season and on the locality in the intertidal.
• 2.2. Anaerobic energy production was only found: (a) in animals from sediments, which fall dry for several hours; (b) in summer and autumn, but not in winter and spring.
• 3.3. A correlation between the extent of anaerobic energy production and the development of gametes was demonstrated.
• 4.4. The process of spawning represents a great stress to the animals. At this time the ability of Arenicola marina to survive anaerobic conditions was reduced drastically.