Download 271 Advances in Environmental Biology, 4(2): 271-276, 2010 ISSN 1995-0756

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Advances in Environmental Biology, 4(2): 271-276, 2010
ISSN 1995-0756
© 2010, American-Eurasian Network for Scientific Information
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Effect of temperature on phagocytosis activity in garden snails Helix aspersa
Ahmad. K. Ma'aqbeh
Department of Biology Al-Hussein bin Talal University Ma'an, Jordan
Ahmad. K. Ma'aqbeh, Effect of temperature on phagocytosis activity in garden snails Helix aspersa;
Adv. Environ. Biol., C(): CC-CC, 2010
ABSTRACT
Helix aspersa, known as garden snail, is a species of land snail, a pulmonate gastropod. This snail is very
common and widespread in Mediterranean region and western Europe. This species is characterized by a
spherical in shape shell with a short spire and a rough surface. The shell color is pale brown, or yellow with
a number of broken dark bands, which give the shell it, is blotchy appearance. This study investigates the
effect of temperature on phagocytosis activity in garden snails H. aspersa. Low and high temperatures depressed
phagocytosis activity and decrease the number of yeast cells phagocyted by H.aspersa hemocytes. The
phagocytosis activity at 25Co is (50.8±4.5), 35 Co (19.2 ± 3.9), 40 Co (3.6±0.89), and 15 Co (4.4±1.1).Two types
of hemocytes cell GLC (Granulocyte like Cell), and HLC (Hyalinocyte like Cell) are diagnosed
Key words: phagocytosis, HLC, GLC, H. aspersa, stress, yeast
Introduction
Invertebrates are widely distributed and can be
found in almost any kind of habitat. Their dispersal
and survival depend on successful defenses
mechanism against various kinds of microorganisms
and parasites, especially that they live in the same
environmental conditions [1].
Invertebrates lack immunoglobulin antibodies and
their associated adaptive immune responses; instead,
they have highly effective innate immune reactions
that defend them against a broad spectrum of
microorganisms [3]. However, previous study has
shown that a specific memory that consider as
acquired immunity in defense systems of
invertebrates [4].
Invertebrate innate immune system is composed
of both cellular and humoral immune responses.
hemocytes-mediated immune responses such as
phagocytosis, nodule formation and melanotic
encapsulation are major cellular responses [5, 6]
Hemocytes from bivalve mollusks have most
often been divided into two types, hyalinocytes and
granulocytes, both of which are phagocytic [7]. These
two cell types have been found in many bivalve
species [7, 8]. In many of these species, granulocytes
have been further subclassified as eosinophilic,
basophilic and neutrophilic cells [9]. Granulocytes
have the ability to phagocytose microbial pathogens
and they contain a mixture of hydrolytic enzymes
that contribute to intracellular killing. They are often
more active phagocytes than hyalinocytes.
Hyalinocytes are smaller than granulocytes. Some
contain few granules and are usually more
morphologically heterogeneous than granulocytes.
Hyalinocytes have been divided into two types, small
hyalinocytes, with large nuclei and scanty cytoplasm,
and large hyalinocytes with low nucleus: cytoplasm
ratios [10].
Phagocytic blood cells are major component of
innate defense in both invertebrates and vertebrates,
where they are also critical for linking innate to
adaptive immunity. Moreover, phagocytosis seems
relevant for one of the most promising candidates for
specific immunity in insects and crustaceans,the
alternatively spliced Down syndrome cell adhesion
Corresponding Author
Ahmad. K. Ma'aqbeh, Department of Biology Al-Hussein bin Talal University Ma'an, Jordan
Tel. +962785656776,
E-mail: [email protected]
[email protected]
Adv. Environ. Biol., 4(2): 271-276, 2010
molecule (Dscam).previous study demonstrate that,
enhanced phagocytosis resulting from prior exposure
to endotoxin or live pathogenic bacteria in
invertebrates [11].
Both the density and functions of mollusc
hemocytes can be affected by changes in
environmental conditions. Different types of stressors,
including pathogens, parasites and xenobiotic, have
been identified as hemocytes effectors. Hemocytes
response patterns may depend on the nature of
stressor [12, 13].
Hemocytes also serve a role in the molluscan
stress response involving endocrine molecules shared
with the vertebrates [14, 15]. Recent studies have
shown that molluscs possess a primitive form of
neuroendocrine response to stress involving
catecholamine and neuropeptides such as
adrenocorticotropic hormone [16].
The objective of this study to investigated the
effect of different temperature on phagocytosis
activity in Helix aspersa
2. Materials and method
2.1. Snails:
Adult specimens of Helix aspersa were obtained
from the northern area of AL-Karak city.it is
governorate lies 140 km to the south of Amman on
the King's Highway. It is situated on a hilltop about
1000 meters above sea level and is surrounded on
three sides by a valley and from Karak can be seen
wonder view of the Dead Sea, and the geographical
coordinates to karak Governorate are 31° 11' 5"
North, 35° 42' 17" East. The snails were kept in
plastic container, at room temperature. The water and
soil was changed every week and the snails were fed
lettuce twice weekly.
Treated snails were kept and incubated in plastic
container at different temperature (25Co, 35 Co, 40
Co, and 15 Co) for two days.
2.2. Hemolymph sampling and counts
Hemolymph was collected directly from the heart
using 22-gauge needles fitted to 3-ml syringes When
the snail retracts into its shell, a drop of hemolymph
was extruded through the hemal pore, and can be
collected using a micropipette. Hemolymph (10 µl)
was directly dropped in hemocytometer. Total
number of living hemocytes and the number of
hemocytes in each cell population identified
according to, Van der Knaap [17] using Optika B350
(Italy) light microscopy.
2.3. Cells analysis and morphology
2.3.1. Live cell analysis
272
The analysis of live hemocytes was carried out
by placing a 30 µl of whole hemolymph on acid
alcohol cleaned microscope slides and allowing the
cells to adhere for 10 min. The slides were then
covered with a glass cover slip and viewed at high
magnification with an Inverted phase microscope
(Leica, Germany)
2.3.2. Staining method
After adhesion to slides, hemocytes were fixed
for 20 min in methanol (80%) and then immersed in
Giemsa stain for 30 min before being washed in
distilled water and air dried. Slides were observed by
Optika B350 (Italy) light microscopy.
2.4. Phagocytic activity
Phagocytic activity was assessed using baker’s
yeast, Saccharomyces cerevisiae (Sigma Aldrich), as
target cells. Five milligrams of yeast was suspended
in 5ml distilled water and mixed with an equal
volume of Congo red. The suspension was autoclaved
at 120 Co for 15 min before being washed twice by
centrifugation at 1300g for 5 min and resuspended in
10 ml distilled water .To measure phagocytosis; 100
ml of whole hemolymph (adjusted to 1×106
hemocytes ml-1) was placed on glass coverslips
(22×22 mm) and incubated in a moist chamber.
Hemocytes were allowed to settle and adhere for 20
min at room temperature (25 Co). The supernatants
were then removed and the adherent hemocytes were
rinsed 5 times with distilled water. The coverslips
were overlaid with 100 ml of Congo red stained
yeast (0.7×106 ml-1) and incubated for 30 min at
room temperature. The coverslips were washed 10
times with distilled water to remove nonphagocytosed yeast cells. They were then inverted
over clean microscope slides and sealed with nail
polish. A minimum of 100 hemocytes on each
coverslip were examined and the number of
hemocytes that had phagocytosed one or more yeast
was recorded so that the percentage of phagocytic
cells could be calculated [10].
2.5. Statistical analysis
All data were presented as means±standard
errors. All experiments were conducted three times.
Data are shown for one representative experiment
from each trial. In each experiment, different groups
of five snails were tested at each time point. Data
were analyzed using the Microsoft Excel package.
Statistical analysis was performed by Student’s t-test
to determine the significance of differences between
mean values. Differences were considered to be
significant if P<0.05[10]
Adv. Environ. Biol., 4(2): 271-276, 2010
3. Results
3.1. Hemocyte morphology
Two major hemocytes types were identified by
light microscopy according to presence or absence of
granules in their cytoplasm, hyalinocytes-like cells
(HLC) and granulocytes-like cells (GLC). HCL were
the first type of hemocytes identify in H. aspersa
(Fig. 1B) these cells comprised 57.6 ±2.6% of the
total hemocytes population. They varied in size
within a range of 6–10µm, with an average diameter
of 9 ± 0.9 µm. HLC had an amoeboid movement
and they formed filopodia. HLC were also
characterized by a cytoplasm containing no granules.
Some HLC maintained a round shape when attached
to glass slides and spread only slightly, forming some
small filopodia. GLC were the second type of
hemocytes identified in H. aspersa (Fig.1A). These
cells comprised 42.4 ± 2.6% of the total hemocytes
population. They varied in size from 5–12 µm with
an average diameter of 9.6 ± 1.4 µm. GLC adhered
to glass slides within 15–20 min and they were
highly ameboid after they adhered. They also had the
ability to form many long filopodia, and were
characterized by numerous cytoplasmic granules.
3.2. Phagocytosis activity
H.aspersa hemocytes were capable of ingesting
yeast cells (Fig.2A). The phagocytic activity was
measured at different temperature (Fig.2B), and a
significant decreased in activity of H.aspersa held at
35 Co (19.2 ± 1.95) as compared to those treated at
25 Co as control 50.8±2.3,( P < 0.05),and the activity
represent more decreasing at 40 Co (3.6±0.4) (P <
0.05) than the activity at 35 Co as compared to the
activity at 25Co . When the H.aspersa kept in low
temperature (15Co), the phagocytic activity dropped
to (4.4±0.6) with compared to those at 25Co. very
little difference in phagocytic activity was found
between H.aspersa kept at 40 Co and those kept at
15 Co. The average number of yeast cells which was
detected in haemocyte of H.aspersa kept at 25Co is
(4cells±0.4), 35Co (2cells±0.4), 40 Co (1.2cells±0.2),
and 15 Co (1.2cells±0.2) (Fig.2 C).
4. Discussion
Hemocyte classification in invertebrates is often
a controversial issue. Simply because there is no
universal procedure to classify hemocytes [10]. In
bivalves mollusks, hemocytes have been classified in
many species, such as R. philippinarum, M. lusoria,
C.gigas, C. farreri, T. crocea, D. polymorpha, and
M. galloprovincialis [18, 19]. Hemocytes
classification depends mainly on host defense,
including the types of granules and enzymes found
273
within cells [10].
Blood cells are differentiated into two types:
granulocytes and hyalinocytes, [7, 8]. The results of
the present study confirm that there are two types of
hemocytes (type I, type II), this result are similar to
that reported in helix aspersa maxima into(type I,
type II). [20]
Similar finding has been reported earlier in P.
corneus [21, 22]. Haemocytes type I in H. aspersa
morphologically similar to granulocytes, and
haemocytes II to hyalinocytes therefor it is possible
to suggest that hemocyte type I is Granulocyte like
Cell (GLC), and hemocyte type II is Hyalinocyte like
Cell (HLC).
Several studies have demonstrated that defence
mechanisms in mollusks are susceptible to changes in
environmental factors, such as temperature, salinity,
dissolved oxygen, food availability and presence of
pollutants [23, 24, 31, 32, 33]. In particular, increases
in temperature have been shown to induce mortality
in bivalves, probably due to fact that high
temperature induced suppression to the immune
system in these animals [25, 34]. Furthermore, the
data obtained of the present study demonstrated that
H.aspersa hemocytes are sensitive to temperature and
there is a good correlation between temperature and
phagocytic activity in this species.
Phagocytosis is a temperature-dependent process
in bivalve molluscs, so it is the main mechanism
against foreign materials in snail's internal defence
system [26]. In the present study, a significant
decrease of phagocytic activity was found in snails
kept at (15 Co, 35Co, 40 Co) with respect to those
kept at 25 Co. Similar finding has been reported in
C. virginica when exposed to a temperature variety
for 1 week [27]. Conversely, Carballal MJ [28] found
that the percentage of phagocytic haemocytes from
M. galloprovincialis was lower at 10 Co than at 20
Co and 30 Co. [29] observed no differences between
phagocytosis at 15 Co and 21 Co in both, R.
decussatus, and M. galloprovincialis. In the present
study, the efficiency of hemocyte for phagocyted
yeast cell decreased at 15 Co to (1.2 cells), 35 Co to
(2 cells), and 40 Co to (1.2 cells) respectively when
compared with 25 Co (4 cells), a previous study [30]
indicate that the max number zymosan particles was
phagocyted by hemocytes cells is (3 particles) at
room temperature.
In conclusion, the influence of temperature on
activity of phagocytosis has been investigated, and
showed that both high temperature and low
temperature have a clear effect on phagocytosis. As
the temperature increased, the activity is clearly
reduced. A similar pattern has been recorded with
low temperature. Two types of cells (GLC, HLC)
seem to have role in phagocytosis in the present
investigation.
Adv. Environ. Biol., 4(2): 271-276, 2010
274
Fig. 1. Light microscope of H.aspersa hemocytes at (40X magnification power). (A) Granulocyte-like cell,
(B) Hyalinocyte-like cell.
Fig 2A: Hematocytes cells ingested the yeast cells scanned light microscope at 40X. B: the effect of
temperature in phagocytosis activity in H.aspersa snails C: the effect of temperature in number of
yeast cells phagocytic by H.aspersa snail's hemocytes
Acknowledgments
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I thank prof. Khalil Altaif and Dr. Saleem
Adaileh for her help, and biology department in ALHussein bin Talal University for providing the
chemicals and devices and for her help.
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