298
G. La Spada et al.
ing cellular elements of various types still organized in tissue fragments and, after shaking, isolated, capable of reaggregation. Alternatively,
Salleo et a1. (1996) and Barra et a1. (1997) did isolate acontial nematocytes of Aiptasia diaphana,
treating the tissue with an SCN- isotonic solution. Thus isolated, the nematocytes appeared
anatomically intact, but lacking in a
mechanosensorial apparatus. and after being
exposed to saline hypotonic solutions (35%),
showed a capacity to regulate cell volume (La
Spada et a!. 1999), a feature which is vital in order
to maintain cell volume constant in conditions of
hyposmotic or hyperosmotic shock (respectively, Regulatory Volume Decrease RVD or
Regulatory Volume Increase RVI). The mechanisms of regulation of volume are due to outflow
of ions such as K+ and Cl-, to the H+ /K+
exchange. to the cotransport of K+ and CI-, or to
the Na+/Ca 2 + exchange, and to the extrusion of
amino acids. The cause of this is, in many cases,
an increase in intracellular concentration of
Ca 2 +, either flowing from outside through
stretch-activated ion channels, or being released
from intracellular stores. A dissertation on the
mechanisms of cell volume regulation has been
recently delivered by Lang et a1. (1998). Volume
regulation was investigated on cells of Mammals,
Amphibians and Crustacea, but not on cells of
Coelenterates (Morris 1990). In the present
investigation, we observed the effects of isolation
on the morphological and functional characteristics of nematocytes isolated from Aiptasia
diaphana, an Anthozoan which lives in the
euphotic zone of the brackish pond Faro
(Messina, Italy), the salinity of which can vary
for both anthropical and climatic factors suggesting the existence of osmoregulatory mechanism in species living there. We therefore decided to compare the morphological features of the
isolated nematocytes both by heat dissociation
as a physical method, and by treatment with
SCN- as a chemical method, evaluating morphological features by observation with scanning
electron microscopy (SEM), and functional features by measuring the changes in volume of the
nematocytes under hyposmotic stress of 35%.
Materials and Methods
Nematocytes were isolated from tentacles of
Aiptasia diaphana collected in the brackish
pond Faro (Messina), maintained in a closed-circuit aquarium at 19°-24 D C and fed with prawn
meat. The tentacles were excised using ophthalmic scissors from animals previously anaesthetized with a solution of MgC1 2 0.6 M and artificial sea water (ASW) in a 1:1 ratio. The composition of ASW (in mM) used in all tests was NaCI
520, KCl 9.7, MgC1 2 24, MgS04 28, CaCl 2 10, imidawl 5, pH 7.6, n= 11 00 mOsm/kg. N ematocytes
were isolated either by tissue heat dissociation,
or by treatment with an isosmotic solution (605
mM of SCN- containing 0.01 mM of Ca2+). In
order to obtain isolation by heat dissociation
two different procedures were adopted: the first
consisted of the incubation of tentacles in ASW
rendered hypertonic by adding mannitol
(n=2200 mOsmlkg). The second consisted of
incubation in an isotonic ASW. Tentacles were
incubated in 0.3 ml of ASW at 40°C, or alternativelyat 45°C in a thermostat-regulated double
boiler for either 10 or 20 min. The test-tube was
then returned to room temperature and delicately shaken manually, until the suspension
appeared cloudy. A drop of the obtained suspension was then put on to a slide previously treated with polylysine (1% in distilled water). After a
few minutes, in order to allow sedimentation of
cellular elements, the medium was carefully
absorbed using blotting paper, with accuracy
being checked under the microscope. The hypertonic ASW used to incubate the suspension was
replaced with isotonic ASW. in order to re-establish normal physiological conditions. The elements obtained, which included nematocytes,
nematocysts and other cells, were counted using
a Leitz-Dialux light microscope with an incorporated video-camera nVC mod. TK-1180 E).
About 200 elements were observed, almost
exclusively nematocytes and nematocysts,
immediately after isolation and at one hour
intervals.
Isolation was obtained by the chemical
method, as described by Salleo et a1. (1996) and
Barra et al. (1997). In short, the tentacles, after
being washed several times with ASW Ca2+ 0.01
mM, were treated with a solution of SCN- and
Ca 2 + 0.01 mM for a time not exceeding 5 min;
this caused a contraction of the tentacle, followed
by extrusion of nematocytes from the tissue. The
solution was gradually replaced, first by ASW
Ca 2 + free and then ASW, to restore control conditions. The cells isolated by the two methods were
then maintained in a damp room at
G. La Spada et al.
ing cellular elements of various types still organized in tissue fragments and, after shaking, isolated, capable of reaggregation. Alternatively,
Salleo et a1. (1996) and Barra et a1. (1997) did isolate acontial nematocytes of Aiptasia diaphana,
treating the tissue with an SCN- isotonic solution. Thus isolated, the nematocytes appeared
anatomically intact, but lacking in a
mechanosensorial apparatus. and after being
exposed to saline hypotonic solutions (35%),
showed a capacity to regulate cell volume (La
Spada et a!. 1999), a feature which is vital in order
to maintain cell volume constant in conditions of
hyposmotic or hyperosmotic shock (respectively, Regulatory Volume Decrease RVD or
Regulatory Volume Increase RVI). The mechanisms of regulation of volume are due to outflow
of ions such as K+ and Cl-, to the H+ /K+
exchange. to the cotransport of K+ and CI-, or to
the Na+/Ca 2 + exchange, and to the extrusion of
amino acids. The cause of this is, in many cases,
an increase in intracellular concentration of
Ca 2 +, either flowing from outside through
stretch-activated ion channels, or being released
from intracellular stores. A dissertation on the
mechanisms of cell volume regulation has been
recently delivered by Lang et a1. (1998). Volume
regulation was investigated on cells of Mammals,
Amphibians and Crustacea, but not on cells of
Coelenterates (Morris 1990). In the present
investigation, we observed the effects of isolation
on the morphological and functional characteristics of nematocytes isolated from Aiptasia
diaphana, an Anthozoan which lives in the
euphotic zone of the brackish pond Faro
(Messina, Italy), the salinity of which can vary
for both anthropical and climatic factors suggesting the existence of osmoregulatory mechanism in species living there. We therefore decided to compare the morphological features of the
isolated nematocytes both by heat dissociation
as a physical method, and by treatment with
SCN- as a chemical method, evaluating morphological features by observation with scanning
electron microscopy (SEM), and functional features by measuring the changes in volume of the
nematocytes under hyposmotic stress of 35%.
Materials and Methods
Nematocytes were isolated from tentacles of
Aiptasia diaphana collected in the brackish
pond Faro (Messina), maintained in a closed-circuit aquarium at 19°-24 D C and fed with prawn
meat. The tentacles were excised using ophthalmic scissors from animals previously anaesthetized with a solution of MgC1 2 0.6 M and artificial sea water (ASW) in a 1:1 ratio. The composition of ASW (in mM) used in all tests was NaCI
520, KCl 9.7, MgC1 2 24, MgS04 28, CaCl 2 10, imidawl 5, pH 7.6, n= 11 00 mOsm/kg. N ematocytes
were isolated either by tissue heat dissociation,
or by treatment with an isosmotic solution (605
mM of SCN- containing 0.01 mM of Ca2+). In
order to obtain isolation by heat dissociation
two different procedures were adopted: the first
consisted of the incubation of tentacles in ASW
rendered hypertonic by adding mannitol
(n=2200 mOsmlkg). The second consisted of
incubation in an isotonic ASW. Tentacles were
incubated in 0.3 ml of ASW at 40°C, or alternativelyat 45°C in a thermostat-regulated double
boiler for either 10 or 20 min. The test-tube was
then returned to room temperature and delicately shaken manually, until the suspension
appeared cloudy. A drop of the obtained suspension was then put on to a slide previously treated with polylysine (1% in distilled water). After a
few minutes, in order to allow sedimentation of
cellular elements, the medium was carefully
absorbed using blotting paper, with accuracy
being checked under the microscope. The hypertonic ASW used to incubate the suspension was
replaced with isotonic ASW. in order to re-establish normal physiological conditions. The elements obtained, which included nematocytes,
nematocysts and other cells, were counted using
a Leitz-Dialux light microscope with an incorporated video-camera nVC mod. TK-1180 E).
About 200 elements were observed, almost
exclusively nematocytes and nematocysts,
immediately after isolation and at one hour
intervals.
Isolation was obtained by the chemical
method, as described by Salleo et a1. (1996) and
Barra et al. (1997). In short, the tentacles, after
being washed several times with ASW Ca2+ 0.01
mM, were treated with a solution of SCN- and
Ca 2 + 0.01 mM for a time not exceeding 5 min;
this caused a contraction of the tentacle, followed
by extrusion of nematocytes from the tissue. The
solution was gradually replaced, first by ASW
Ca 2 + free and then ASW, to restore control conditions. The cells isolated by the two methods were
then maintained in a damp room at