Calcium fluxes in single muscle fibres measured with a glass scintillator probe. (1/218)

1. An intracellular glass scintillator (Caldwell & Lea, 1973) has been used to obtain a continuous record of the influx of 45Ca into single muscle fibres of the barnacle, Balanus nubilus. 2. In the presence of intracellular EGTA (final concentration greater than 3 mM/kg), the scintillator detected an initial fast phase to the influx (half-time = 18.3 min, compartment size = 4.1% fibre volume) followed by a slow, linear phase which gave a value for the Ca influx of 1.2 p-mole . cm-2 . sec-1. The efflux of 45Ca was also measured with the scintillator by transferring a 45Ca-loaded fibre into 45Ca-free saline. Two exponential phases of efflux were detected with half-times of 16.2 and 500 min. 3. The characterisitics of the fast phase of the influx and efflux are similar to those of the influx of the impermeant sucrose and inulin, suggesting that the fast phase represents exchange with the extracellular 'cleft space'. This phase was insensitive to external La3+ (2 mM). 4. The slow phase is considered to represent the flux of Ca across the surface membrane. It was inhibited by external La3+ (2 mM) and stimulated by replacing external Na+ with Li+. 5. When EGTA-injected fibres were depolarized using an axial, intracellular electrode the Ca influx, measured from the slow phase, was increased. At higher concentrations of intracellular EGTA (6--22 mM/kg), the extra Ca influx due to a rectangular, depolarizing current pulse was proportional to the number of Ca spikes it produced. A single Ca spike gave an extra Ca influx of 19--48 p-mole . cm-2. External D600 (5 x 10(-4)M) inhibited both Ca spike and the extra Ca influx. 6. At lower intracellular EGTA concentrations (3.6--11 mM/kg), a 50 mV depolarization of 250 msec duration gave a mean extra Ca influx of 80 p-mole . cm-2. The upper value was 145 p-mole . cm-2 and this would increase the total internal Ca by 4.1 micrometer/kg. It is calculated that if all this extra Ca was bound to the myofibrillar sites for tension, it would only produce 6.2% of the force expected for a similar depolarization in a fibre with no intracellular EGTA.  (+info)

Environmental heterogeneity and balancing selection in the acorn barnacle Semibalanus balanoides. (2/218)

The northern acorn barnacle Semibalans banlanoides occupies several intertidal microhabitats which vary greatly in their degree of physical stress. This environmental heterogeneity creates distinct selection regimes which can maintain genetic variation in natural populations. Despite considerable attention placed on the link between spatial variation in fitness and balancing selection at specific loci, experimental manipulations and fitness estimates for molecular polymorphisms have rarely been conducted in the wild. The aim of this transplant experiment was to manipulate the level of physical stress experienced by a cohort of barnacles in the field and then investigate the spatial variation in fitness for genotypes at three loci: two candidate allozymes and the mitochondrial DNA control region. The viability of mannose-6-phosphate isomerase (Mpi) genotypes was dependent on the level of physical stress experienced in the various treatments; alternative homozygotes were favoured in alternative high stress-low stress environments. In contrast, the fitness of genotypes at other loci was equivalent among treatments and unaffected by the manipulation. Evaluated in the light of balancing selection models, these data indicate that the presence of multiple environmental niches is sufficient to promote a stable Mpi polymorphism in barnacle populations and that allelic variation at this locus reflects the process of adaptation to the heterogeneous intertidal landscape.  (+info)

Stimulation by aldosterone of the sodium efflux in barnacle muscle fibres: effects of RNA inhibitors and spironolactone. (3/218)

1. Single muscle fibres from the barnacle Balanus nubilus have been studied to provide information about the mode of action of aldosterone on Na transport in a symmetric cell. 2. Neither internal nor external application of aldosterone modified the course of the Na efflux. However, fibres pre-exposed overnight to a dose of aldosterone showed a biphasic response to external aldosterone. The first phase was prompt in onset and consisted of a falling rate constant for Na efflux becoming a constant. This has been interpreted as indicating that aldosterone acts by releasing sequestered or bound Na. The second phase was delayed in onset with the average latent period being 68 min. It consisted of a step-up in the rate of Na efflux, followed by a transitory rise in the efflux rate constant. This second phase was dose-dependent, the minimum concentration being 10(-9) M. 3. Internal application of aldosterone in a concentration as low as 10(-10) M promptly stopped the rate constant for Na efflux from further falling but failed to cause delayed stimulation. 4. The response of the Na efflux into Li-ASW following external application of aldosterone was bi-phasic. The magnitude of the delayed stimulation was comparable to that found in controls. 5. No correlation between latent period or size of the internal Na bound fraction, and the magnitude of the delayed stimulation could be established. 6. Internal application of actinomycin-D, alpha-amanitin or cordycepin shortly before application of external aldosterone caused complete abolition of the delayed stimulation. 7. Overnight pre-exposure of the barnacle to actinomycin-D caused complete abolition of the falling rate constant for Na efflux, as well as the delayed stimulation caused by external aldosterone. 8. Internal application of spironolactone SC-14266 shortly before external application of aldosterone caused complete abolition of the biphasic response to the steroid. 9. It is concluded that (i) barnacle fibres can be made sensitive to aldosterone, (ii) the biphasic effects on the Na efflux depend on prior induction of RNA, (iii) the first effect caused by internal or external aldosterone involves mobilization of sequestered Na, (iv) the results obtained with spironolactone are in keeping with the current view that aldosterone interacts with a receptor system before causing de-repression. 10. The implications of the finding that aldosterone releases sequestered Na are briefly touched upon.  (+info)

Barnacle cement proteins. Importance of disulfide bonds in their insolubility. (4/218)

Barnacles produce a cement that is a proteinaceous underwater adhesive for their secure attachment to the substratum. The biochemical properties of the cement have not previously been elucidated, because the insolubility of the cement proteins hampers their purification and characterization. We developed a non-hydrolytic method to render soluble most of the cement components, thereby allowing the proteins to be analyzed. Megabalanus rosa cement could be almost completely rendered soluble by its reduction with 0.5 m dithiothreitol at 60 degrees C in a 7 m guanidine hydrochloride solution, the high concentration of dithiothreitol being indispensable to achieve this. The effectiveness of this reduction treatment was confirmed by the detachment of the barnacle from the substratum. Three proteins comprising up to 94% of the whole cement were identified as the major cement components. The cDNA clone of one of these major proteins was isolated, and the site-specific expression of the gene in the basal portion of the adult barnacle, where the cement glands are located, was demonstrated. A sequence analysis revealed this cement component to be a novel protein of 993 amino acid residues, including a signal peptide. This is the first report of the major component of the barnacle cement protein complex.  (+info)

Effect of inducers and inhibitors on the expression of bcs genes involved in cypris larval attachment and metamorphosis of the barnacles Balanus amphitrite. (5/218)

We examined the expression of six barnacle cypris larva-specific gene (bcs) cDNAs (bcs-1, -2, -3, -4,- 5, and -6), the bcs genes, by using Northern blot analysis under various conditions that induced or inhibited cypris larval attachment and metamorphosis. Inducers of larval attachment and metamorphosis, such as a neurotransmitter, tended to increase the expression of bcs mRNAs. All inhibitors of larval attachment and metamorphosis, such as G protein-coupled receptor agonists/antagonists, inhibitors of tyrosine kinase-linked receptors and inhibitors of their signal transduction, suppressed the expression of bcs-6 mRNA alone, but affected differentially other bcs genes. These results strongly suggest that the bcs-6 product plays a key role in triggering the attachment and metamorphosis of cypris larvae into juvenile barnacles. The roles of four late bcs genes (bcs-3,-4, -5 and -6) are discussed.  (+info)

Novel barnacle underwater adhesive protein is a charged amino acid-rich protein constituted by a Cys-rich repetitive sequence. (6/218)

Barnacle cement is an underwater adhesive that is used for permanent settlement, and is an insoluble protein complex. A method for rendering soluble the cement of Megabalanus rosa has been developed, and three major proteins have been identified in a previous study. To survey the M. rosa cement proteins in a lower molecular mass range, the cement proteins were separated by reversed-phase HPLC and a previously unidentified protein named 20 kDa M. rosa cement protein (Mrcp-20k) was found. Mrcp-20k cDNA was cloned to reveal its primary structure. This cDNA was 902 bp long and encoded a 202 amino acid-long open reading frame, including 19 amino acids of the signal sequence. The molecular mass in the disulphide form was calculated to be 20357 Da and the isoelectric point of the mature polypeptide was 4.72. Mrcp-20k was characterized by an abundance of Cys residues and charged amino acids. The most common amino acid was Cys (17.5%), with Asp (11.5%), Glu (10.4%) and His (10.4%) following in order of magnitude. The alignment of the Cys residues indicated the primary structure of this protein to consist of six degenerated repeats, each about 30 residues long. Mrcp-20k has no intermolecular disulphide bonds and no free thiol groups of Cys in the insoluble cement complex. Abundant Cys is thought to play a role in maintaining the topology of charged amino acids on the molecular surface by intramolecular disulphide-bond formation. The possible function of abundant charged amino acids, including the interaction with a variety of surface metals on the substratum, is discussed.  (+info)

Host-associated speciation in a coral-inhabiting barnacle. (7/218)

Host specificity of symbionts is considered an important factor associated with sympatric speciation. Here, we examine host specificity and the degree of host-associated speciation in the barnacle Savignium milleporum, an obligate symbiont of the hydrocoral Millepora (the "fire coral"). Little morphological variability was revealed between barnacles collected from two morphs of the hydrocoral Millepora dichotoma (encrusting or branching) or from its congener Millepora platyphylla, but a molecular analysis revealed an unexpected pattern of DNA sequence divergence. The sequences of the 12S mitochondrial rDNA were nearly identical within each of the three barnacle populations (average sequence divergence <1%), and the sequences obtained for barnacles collected from the two different morphs of M. dichotoma differed considerably (ca. 9% average sequence divergence). However, S. milleporum collected from M. platyphylla were nearly identical to the barnacles from the branching M. dichotoma (<0.5% average sequence divergence). The pattern of speciation demonstrated by Savignium barnacles indicates the gradual colonization of similar hosts (i.e., sequential evolution), rather then "casual" colonization, as indicated for other systems. If this is indeed so, then symbiont phylogeny should roughly correlate with host phylogeny. Additionally, the data support the "rendezvous host" hypothesis, which invokes the opportunity of both sexes to meet as a major component for which selection favors the costly habit of host specificity.  (+info)

Spectral correlates of a quasi-stable depolarization in barnacle photoreceptor following red light. (8/218)

1. Illumination of B. eburneus photoreceptors with intense red light produces a membrane depolarization that persists in darkness. This quasistable depolarization (latch-up) can be terminated with green light. The phenomenon was investigated with electrophysiological, spectrochemical, and microspectrophotometric techniques. 2. Latch-up was associated with a stable inward current in cells with the membrane potential voltage-clamped at the resting potential in darkness. The stable current could only be elicited at wave-lengths greater than 580 nm. 3. Light-induced current (LIC) was measured at various wave-lengths in dark-adapted photoreceptors with the membrane voltage-clamped to the resting potential. The minimum number of photons required to elicit a fixed amount of LIC occurred at 540 nm, indicating that the photoreceptor is maximally sensitive to this wave-length of light. The photoreceptor was also sensitive to wave-lengths in the near-U.V. region of the spectrum (380-420 nm). 4. Steady red adapting light reduced the magnitude of the LIC uniformly at all wave-lengths except in the near-U.V. region of the spectrum; sensitivity was reduced less in this region. 5. The spectrum for termination of the stable inward current following or during red light was shifted to the blue (peak about 510 nm) compared to the peak for LIC (peak about 540 nm). 6. Absorbance of single cells prepared under bright, red light decreased maximally at 480 nm following exposure to wave-lengths of light longer than 540 nm. 7. A pigment extract of 1000 barnacle ocelli prepared under dim, red light had a maximum absorbance change at 480 nm when bleached with blue-gree light. 8. There was no evidence in the latter two experiments of photointerconversion of pigments with absorbance maxima at 480 and 540 nm. Rather, the maximum absorption of the bleaching products seemed to occur at wave-lengths shorter than 420 nm. 9. Since latch-up induction occurs at wave-lengths longer than 580 nm, it may depend on the 540 pigment or on an undetected red absorbing pigment. 10. A photolabile pigment at 480 nm correlated most closely with termination of the stable inward current associated with latch-up.  (+info)

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