The limpet Patella vulgata L. at night in air: effective feeding on Ascophyllum nodosum monocultures and stranded seaweeds

Opportunistically, the limpet Patella vulgata will switch frommicrophagous grazing to feeding on attached macroalgae or, anovel finding, on accumulations of stranded seaweeds. Macroalgalfood is ingested primarily at night during emersion, when thethalli come to lie on the rock surface and are sufficientlydamp. Monocultures of Ascophyllum nodosum are not immune tothe destructive effects of limpets as previously thought. Patellavulgata can graze them down to complete disappearance, thuspushing Ascophyllum back to refuges where P. vulgata is absentor rare. Such refuges were found along a wide range from verysheltered to strongly wave-exposed rocky shores in Brittany,France. By a combination of its macrophagous and microphagousfeeding habits, P. vulgata can control mid-tidal macroalgalassemblages in a similar fashion to the action of sea urchinsupon kelp communities inhabiting the lower intertidal and shallowsublittoral zones of rocky shores. (Received 30 October 2006; accepted 25 May 2007)     

Purification and partial characterization of a carboxypeptidase from the limpet (Patella vulgata)

A carboxypeptidase A-like enzyme has been purified to apparent homogeneity from commercially available acetone powder from the visceral hump of the limpet, Patella vulgata. A two-step procedure involving affinity chromatography on ?-amino-N-caproyl-d-phenylalanine-Sepharose and gel filtration resulted in a 3000-fold purification with an 80% yield. The enzyme is a single polypeptide chain of M_r = 40,000 and exhibits both peptidase and esterase activities, which are characterized by dramatic excess substrate inhibition. Inhibition studies suggest that a metal ion is required for activity and demonstrate that the affinity label, N-bromoacetyl-N-methyl-l-phenylalanine, and a polypeptide carboxypeptidase inhibitor from potatoes (apparent K_i approx. 2 nm) are effective against the limpet enzyme.     

Changes in membrane properties during in-vitro meiotic maturation of the limpet Patella vulgata

Changes in the membrane properties of the oocyte of the mollusk, Patella vulgata, were analyzed following the induction of meiosis reinitiation by paleopedial ganglia extract or by the weak base ammonia. During maturation it was possible to distinguish between an early phase characterized by an initial hyperpolarization and a late phase consisting of a depolarization which triggers an action potential with a long-term overshoot (20 minutes) of the membrane potential. Major changes in individual ionic permeabilities were studied using both current and voltage clamp conditions. The depolarizing phase appears to depend on decreases in K+ membrane permeability. Finally we observed that the overshoot did not appear to be directly related to germinal vesicle breakdown (GVBD) since it was absent in Na-deprived artificial sea water and could be elicited in the presence of TEA bromide, which did not induce maturation. This last observation suggests that it may result from a change in specific K+ ion permeability due to the possible activation of stretch channels.     

Food supply, grazing activity and growth rate in the limpet Patella vulgata L.: a comparison between exposed and sheltered shores

The limpet Patella vulgata L. is an important microphagous grazer on intertidal rocky shores of north-west Europe, occurring across the wave exposure gradient. Groups of P. vulgata were selected at mid-tide level of two exposed shores and two sheltered, fucoid dominated shores on the Isle of Man, British Isles, and manipulated to equivalent densities and population structure. The level of grazing activity and growth rate were determined over a 1-year period. At the same time, the abundance of epilithic microalgae, measured as the concentration of chlorophyll a, was determined as an estimate of food supply. Microagal abundance showed a seasonal pattern in both exposed and sheltered conditions, with higher levels in winter compared to summer. In both seasons, the microalgal resource was more abundant on the sheltered shore studied. The level of grazing activity in P. vulgata showed a seasonal pattern on the exposed but not the sheltered shores. Averaged over the year, grazing activity on the exposed shores was over double that on sheltered shores. Thus, in sheltered conditions, food supply for limpets was high and grazing activity low; in exposed conditions, food supply was low and grazing activity high. The growth rate of P. vulgata, measured as increase in shell length, showed no significant difference between exposed and sheltered shores. Growth rate was also determined in P. vulgata at natural densities. Although the overall density declined with decreasing exposure to wave action, the density per unit area of grazeable substance was higher in shelter. In these populations, the mean growth rate was over twice as high on exposed compared to sheltered shores.     

The role of cyclins in the maturation of Patella vulgata oocytes.

We have cloned and sequenced the cDNAs encoding Patella vulgata cyclins A and B. The cDNA clones contain an open reading frame of 426 and 408 amino acids respectively, which present similarity with cyclins from other species. Cyclin A and B RNAs are present as polyadenylated and non-polyadenylated RNA in prophase oocytes and are completely polyadenylated in metaphase I. During the first cleavages after fertilization the level of cyclin A and B mRNAs is high and drops when the free swimming stage is reached. Using p13suc1-Sepharose bead precipitation we demonstrate that cyclin synthesis is triggered during maturation and that inhibition of protein synthesis makes the cyclins disappear rapidly from the metaphase I oocytes, which shift to interphase condition. By microinjecting antisense oligonucleotides into metaphase I oocytes, we demonstrate that in vivo ablation of cyclin A and B messengers together gives the same result, whereas microinjection of only one oligonucleotide does not show any effect.     

Metabolic responses of the limpet Patella caerulea (L.) to anoxia and dehydration.

This study examined the metabolic responses of the limpet Patella caerulea (L.) to anoxia and dehydration, attempting to tease apart the effect of these two stressful conditions, which are often not clearly distinguished in experiments. Specimens were exposed to: (a) oxygen-free sea water; (b) oxygen-saturated water (controls); (c) low-humidity air (55% RH); and (d) high-humidity air (100% RH). For each of the treatments, we took samples of five specimens after 6 and 18 h of exposure to the experimental conditions and determined the concentrations in the foot muscle of succinate, acetate, propionate, aspartate and alanine. Exposure to anoxia caused an increase in the levels of succinate (6 and 18 h) and acetate and propionate (18 h) with respect to control specimens. Anoxia also induced a decrease of aspartate and an increase of alanine after both 6 and 18 h. Exposure to both moist and dry air generally had negligible effects on the organic acid levels. Aspartate content increased after 18 h of exposure to moist air. Alanine levels also increased with respect to control values after exposure to air, with dry air having the more pronounced effect. In conclusion, the results of this study suggest that one should be cautious when inferring anaerobic conditions from the simple exposure of intertidal species to air, without strict control of the experimental conditions and actual respiration rates.     

Development of dorsoventral polarity and mesentoblast determination in Patella vulgata

In Patella vulgata the 32-cell stage represents a pause in the mitotic activity prior to the differentiation of the mesentoblast mother cell 3D. At the onset of this stage, the embryo is radially symmetrical. Nevertheless, the plane of bilateral symmetry is indicated as it passes through the macromeres forming the vegetal cross-furrow. From the early beginning of the 32-cell stage, all four macromeres intrude far into the interior and touch the centrally radiating cells of the first quartet of micromeres. The two cross-furrow forming macromeres (3B and 3D) intrude the farthest and come into contact with the greatest number of micromeres. Finally, the contacts are extended significantly and maintained with only one of these macromeres. From that moment, this cell can be called the macromere 3D and the dorsoventral axis is determined. The evolution of the internal cell contacts between the micromeres of the first quartet and the macromeres indicates an essential role of the former in the determination of one of the latter as the mesentoblast mother cell, and thus in the determination of dorsoventral polarity.     

Communication compartments in the ectoderm of embryos of Patella vulgata

Summary Patterns of gap junctional communication in the ectoderm of embryos of Patella vulgata have been studied by intracellular injection of the fluorescent dye Lucifer Yellow, and by analysis of its subsequent spread to adjacent cells (dye-coupling). We found that dye-coupling became progressively restricted to different domains of the ectoderm, forming communication compartments. These communication compartments are characterized by their high coupling abilities within the compartment, and reduction of coupling across their boundaries. During development, the pretrochal (anterior) ectoderm becomes subdivided into two communication compartments, the apical organ and the anlage of the head ectoderm. The posttrochal (posterior) ectoderm becomes subdivided into different communication compartments in two successive phases. Firstly, in the 15-h embryo the dorsal and ventral domains of the ectoderm form separate communication compartments. A dorso-ventral communication boundary restricts the passage of dye between the two domains. Secondly, in the 24-h embryo dye-coupling becomes further compartmentalized in both the dorsal and ventral domains. These compartments correspond to the anlagen of different ectodermal structures. In order to study whether any level of coupling persists between the ectodermal compartments we injected currents through a microelectrode inserted into one cell of one compartment and monitored its spread by means of a second microelectrode inserted into one cell of another compartment (electrical coupling). Despite the absence of dye-coupling, electrical coupling between the ectodermal dye-coupling compartments was detected, which suggests that some level of communication is maintained between compartments. Our results demonstrate that within the ectoderm layer of Patella vulgata the transfer of dyes becomes progressively restricted to communication compartments and, concomitantly with the specification of the different ectodermal anlagen, these compartments become subdivided into smaller communication compartments.     

Activation of polyphosphoinositide metabolism at artificial maturation of Patella vulgata oocytes.

The metabolism of polyphosphoinositides (PPI) has been investigated during the meiosis reinitiation of the oocytes of a prosobranch mollusk, the limpet Patella vulgata. Meiosis reinitiation which leads to germinal vesicle breakdown (GVBD) and metaphase-1 spindle formation was artificially induced by treating the prophase-blocked oocytes with 10 mM NH4Cl, pH 8.2. This treatment, which results in a rise in intracellular pH, triggered a general increase in polyphosphoinositide synthesis. Determinations of phosphorus content showed that maturation induced a 30 to 50% increase in both phosphatidylinositol (PI) and phosphatidylinositol-1 monophosphate (PIP) concentrations. Incorporations of 32PO4 and [3H]inositol have been measured in three classes of polyphosphoinositides: PI, PIP, and phosphatidylinositol 4,5-bisphosphate (PIP2). By comparing incorporation rates of the radiolabeled precursors into PPI before and after meiosis reinitiation, we found that artificial maturation by ammonia induced a 50-fold increase in the turnover of these lipids. No significant burst of inositol 1,4,5-trisphosphate (IP3) was observed after maturation. We suggest that modifications in PPI metabolism occurring at maturation of Patella oocytes might ensure the formation of an important stock of PPI that would be available for the profuse production of IP3, the messenger responsible for the Ca2+ signal at fertilization.     

New polymorphic microsatellite markers for the limpet Patella rustica and cross-priming testing in four Patella species

Eighteen microsatellite markers have been characterized from the Patella rustica genome. An average of 10 alleles per locus and an expected heterozygosity ranging from 0.054 to 0.937 were observed in a sample of 32 wild individuals from Viana do Castelo, Portugal. Distinct cross-priming amplification rates were recovered on four additional Patella species. Three monomorphic P. rustica loci were polymorphic in other Patella species. The microsatellites developed herein could be a useful intraspecific genetic tool to undertake fine population studies in the genus Patella.