Morphological and functional study of the marginal sphincter of the sea anemones Phymactis clematis and Aulactinia marplatensis from intertidal of Mar del Plata, Argentina

It was made the characterization of marginal sphincter to the species Phymactis clematis (Drayton in Dana, 1849) and Aulactinia marplatensis (Zamponi, 1977), from intertidal ecosystem through their morphogical and functional study. The species P. clematis has a circumscript sphincter of palmate type. This muscle is constituted by a mesogloeal axis and several mesogloeal subaxes. Axis as well as subaxes give a support to the endoderm which border is smooth. Aulactinia marplatensis has a circunscript sphincter pinnate type. The axis has a truncated cone shape while in P. clematis the shape is cylindrical on its origin and it is bifurcated at the end. Both species experiments were carried out using the isolated muscles. They were stimulated at increasing KCl concentrations ranging from 20 to 200 mM. The results were analysed in the form of dose-response curves expressed in tension in grams force vs concentration. Contractil force increases in a sigmoid form to increasing KCl concentrations. The correlation between morphology and function and the differences shown in both species would be related to their intertidal distribution.

The column of sea anemone is constituted of muscle fibers and inmediately or a short distance below the margin, the circular muscle sheet is concentrated into a special band: the marginal sphincter (STEPHENSON, 1928).This structure presents characteristics of smooth muscle (PATRONELLI et al., 1998).The sphincter closes the aperture at the top of the column when the disc and tentacles have been retracted.According to the disposition of mesogloeal axis and subaxes the sphincter can be classified as circumscript, circumscript-diffuse or diffuse (MANUEL, 1981).These morphological types may present structural and physiological differences depending on the species located in intertidal or depth environment (PATRONELLI et al., 2004).In the intertidal ecosystem of Mar del Plata (Argentina), sea anemones are distributed in protected, partially protected or exposed areas.The different conditions in these areas such as pressure of the water column or dashing waves promote different responses in the sphincter (PATRONELLI et al., 1987).
The aim of this work is to make a comparative study of the marginal sphincter of both species by morphological and functional analysis.

MATERIAL AND METHODS
Specimens of the sea anemones Phymactis clematis and Aulactinia marplatensis, were collected from the middle littoral in the rocky zone of Punta Cantera, Mar del Plata (38°05'S and 57°38'W), Argentina.The samples, all around the same size (30 mm in basal diameter), to avoid another variable, were caught in autumn-winter, period whereon the responses are optimal to mechanical stimuli, discarding those specimens with typical spawning position (PATRONELLI et al., 2002).Both species were collected in the same area under the same environmental conditions and in different positions on the substrate.Aulactinia marplatensis is distributed on the vertical walls on the rocks, unlike P. clematis whose distribution is extended over plane and horizontal hollows.The organisms were maintained at room temperature in an aquarium with decanted and aerated sea water.They were used between one and seven days after collection.
Morphological study.The sphincter analysis was made by dissection techniques through longitudinal plane of the organism and it was placed in a Petri dish with fixed solution (formaldehyde solution neutralizated 40% or absolute alcoholic solution).The sphincter was observed upon stereoscopic microscope; by objectives of different resolutions the following meristic dates were measured: length and width of sphincter, of mesogloeal axis and mesogloeal subaxes.
Physiological study.Sphincter extraction and preparation were carried out according to the techniques described by PATRONELLI et al., 1987.The variations of tension were measured by a force transducer connected to a polygraph.The muscles were stimulated at different KCl concentrations (20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 mM) and their responses were analysed in the form of dose-response curves expressed in tension in grams force (gr) vs concentration.Data are presented as average values ± standard error of experiments.Statistical analysis was performed using the Student's t test.P values < 0.05 were considered statistically different.

RESULTS
Morphological study.The species P. clematis has a circumscript sphincter of palmate type (Fig. 1) which is between 1.06 to 1.67 mm long and between 0.76 to 1.21 mm wide.This muscle is constituted by a mesogloeal axis and several mesogloeal subaxes.Axis as well as subaxes give a support to the endoderm which border is smooth.The mesogloeal axis is between 0.30 to 1.06 mm long and between 0.15 to 0.60 mm wide.It is possible to count a number of more than 5 subaxes (6-8) which dimensions are between 0.07 to 0.91 mm long and 0.07 to 0.30 mm wide.The species A. marplatensis has a circumscript sphincter pinnate type (Fig. 2) which is between 1.21 to 1.76 mm long and between 1.06 to 1.36 mm wide.This muscle is constituted by a mesogloeal axis and many subaxes.Axis and subaxes give a support to endoderm of sphincter which border is folded.The mesogloeal axis is 0.30 to 0.76 mm long while its width is between 0.60 to 0.91 mm (n= 25).This axis has a truncated cone shape while in P. clematis the shape is cylindrical on its origin and bifurcated at the end.The mesogloeal subaxes are numerous which number is more than 9 and their dimensions are between 0.45 to 1.21mm long and 0.15 mm wide (n= 25).

DISCUSSION
The species Phymactis clematis has a smooth edge sphincter of the circumscript type, which presents constant physiological activity even if it is exposed to environmental dissecation (PATRONELLI et al., 1999).Aulactinia marplatensis is found in the same study area and its sphincter presents folded edges, making a stronger structure.This morphological adaptation might be related to the localization of the species since that it is distributed on the vertical walls on the rocks, unlike P. clematis whose distribution is extended over plane and horizontal hollows where pressure of water is secondrate.
The analysis of sphincter contractility in both species, showed that P. clematis developed a higher contractile force than A. marplatensis for all KCl concentrations.The contractile force to 200 mM KCl was minor.This event might be related to the higher depolarization which leads to decreasing calcium ions influx, because these ions positively charge the intracellular medium repulsing more calcium ions from entering (PATRONELLI et al., 2000).
Comparing sphincters of both species, morphological and functional differences were found, which might be related with their distribution in intertidal.Sphincters of P. clematis populations distributed in exposed areas present great differences compared to specimens from protected environments.The ones which comes from exposed environments have developed sphincters adapted to resist the continuous dashing of waves.Physiological studies correlated well with morphological observations (PATRONELLI et al., 1987).
Instead, in A. marplatensis there was no correlation between morphology and function in the sphincters, as it was expected to obtain a higher contractile force, because these are stronger and have a pinnate disposition.These results suggest that A. marplatensis, due to its localization is subjected to direct wave impact, and its sphincter instead of developing a higher contractile force than in P. clematis uses, as an adaptative strategy, a sustained contraction and a longer relaxation.
As is it described for molluscan smooth muscles, the sphincter maintains a stretch-resistance state for long periods.This phenomenon suggests us that contraction of this smooth muscle produces a "catch" tension.This property is present for the so called "paramyosine muscles" (KRYVI, 1975) among which may be included these sphincters because under biochemical analysis in our laboratory, we have identified a protein with a molecular weight of 106 KDa (data non published) which might correspond to paramyosine.This protein is typical in the thick filament of invertebrates.The sphincter of A. marplatensis might present more quantity of this protein than in P. clematis, which would allow it to combine the ability to provide relatively free movement activity and a firm position for sustained periods.