Vol.1, No.2, 54-60 (2011) Open Journal of Animal Sciences
doi:10.4236/ojas.2011.12007
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
Impact of the photosensitizers hematoporphyrin coated
gold nanoparticles on biomphalaria alexandrina snails
Karem El-Hommossany1, S. A. El-Sherbibni2
1Medical Malacology Department, Theodor Bilharz Research Institute, Giza, Egypt;
2National Institute of Laser Enhanced Scince, Giza, Egypt; Karmelhommossany@yahoo.com
Received 27 April 2011, revised 11 May 2011, accepted 27 June 2011.
ABSTRACT
The present study was done using two concen-
trations of the photosens itizer Hema toporphyrin
coated Gold Nanoparticles (HpdGNP3) (5 × 10–6,
5 × 10–5 mole/dicemeter–3), to evaluate their ef-
ficacy on survival rate, egg-laying capacity of
Biomphalaria alexandrina snails and on histo-
logical deteriorations in their hermaphrodite
gland. B. alexandrina snails were incubated for
12 hours at each tested concentration in the
dark, thereafter they were exposed to direct
sunlight (336.2 W/m2) for either 2 or 4 hours
followed by 24 hours of recovery. Control snails
were treated with these concentrations without
exposure to light irradiance. Another experi-
ment was carried out simultaneously and the
snails were left for 4 weeks of recovery to
evaluate their egg-laying capacity (Mx). The re-
sults indicate that 5 × 10–5 Mdm–3 (HpdGNPs)
with 4 hours of exposure sunlight suppressed
the survival rate of B. alexandrina snails by 50%.
Meanwhile, control snails incubated with 5 ×
10–5 Mdm–3 HpdGNPs were not affected and still
alive (100%). For snail’s fecundity (Mx), treated
snails laid low number of eggs throughout the
recovery period (4 weeks), in comparison with
that of control ones. The highest value of Mx for
snails treated with 5 × 10–5 Mdm–3 Hpd coated
GNPs was recorded at the 3rd week of recovery
period, being 6.7 eggs/snail, compared to 37.6
eggs/control snail. This has a negative reflect
on the reproductive rate (Ro) of treated snails as
it was reduced under these conditions by 76.6%
and 86.1%, respectively.Histological tests re-
vealed injuries in spermatocytes, oocytes, sev-
eral degenerations of B.alexandrina hermaph-
rodite gland then evacuations in many gonad’s
cells which severely suppressed their capacity
for egg-laying. It is concluded from the present
work that exposing B. alexandrina snails to
sublethal concentrations of the photosensiter
Hpd coated GNPs (12 hours incubation, 4 hours
exposure to 336.2 W/m2) significantly reduced
their reproductive capacity that may have a
negative reflect on schistosomiasis transmis-
sion.
Keywords: Biomphala ri a Alexandrina Snails;
Hematoporphyrin Coated Gold Nanoparticles;
Snail’s Egg-Laying Capacity; Histology
1. INTRODUCTION
Schistosomiasis is an important public health prblem
in Egypt and several developing countries (Pasvol and
Hoffman, 2001). Control of this parasitic disease could
be through treatment of infected persons, health educa-
tion, sanitation and snail control. Thus, snail control
strategies are considered a priority for preventing or
minimizing schistosomiasis transmission (Lardans and
Dissous, 1998). These strategies have been based upon
elimination of the snail intermediate hosts chemically,
biologically or environmentaly. However, further tech-
niques are required in this field. So, phtosensitizing
compounds could be evaluating for controlling noxious
water parasites, since sunlight activated dyes character-
ized by a high efficiency, lack of mutagenic activity and
negligible toxicological risk to humans and other mam-
malian species (Ben Amor et al, 1998).
Hematoporphyrin derivatives have been used as phto-
sensitizing agent in the presence of sunlight as well as
artificial light (Spikes, 1984). Later, Abdel-Kader et al
(2001) observed that incubation of Lymnaea natalensis
snails with 103 Mdm3 Hematoporphyrin (Hpd) for 12
hrs, followed by 30 min of exposure to sunlight (300
W/m2) killed these snails after 24 hrs of recovery.
El-Tarky (2005) noticed that mortality rate of adult B.
alexandrina snails exposed for 4 hrs to 438.8 w/m2 of
sunlight after 12 hours of their incubation with 105
K. El-Hommossany et al. / Open Journal of Animal Sciences 1 (2011) 54-60
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
5555
Hpds, was 52%. The author added that the effect of
Hpds against snails increased in the alkaline media (pH
= 8), while it decreased at the acidic ones (pH = 6). It
was stated that porphyrins and their derivatives exhibited
a potent phytotoxic effect against gram-positive bacteria
(Bertoloni et al., 1993). In 2006, El-Sayed and
El-Sherbini recorded that LC50 of Hematoporphyrin
(Hpd) against adult B. alexandrina snails was 5 × 105
M/dm-3 after 12 hours of incubation and 2 hours of irra-
diation at ~ 336 W/m2 in sunlight and at 2 × 105 Mdm3
Hpd, they did not lay eggs. The authors added that infec-
tion rates of these snails with S. mansoni were signifi-
cantly suppressed by their exposure to 2 × 105 Mdm3
Hematoporphyrin (Hpd) either pre- or postmiracidial
exposure, in addition to the significant reduction in cer-
carial production/snails treated with 4 × 105 Mdm3 Hpd.
Moreover, histological examinations showed a sever
damage in the hermaphrodite gland of snails exposed to
the sublethal concentrations of Hpd.
Recentely, Ragheb (2009) stated that incubation of B.
truncatus snails for 24 hrs at 10–4 M/L gold nanoparticls
(GNPs) in dark followed by 2 hours of exposure to 650
W/m2 irradiance (Solar Simulator) resulted in 100%
death of snails. The author added that decreasing GNPs
concentration to 105 M/L significantly reduced the
snail’s reproductive rate (Ro) by 48.3% comp ared to that
of control, and this was partially due to the recorded
damage in hermaphrodite gland cells of treated snails.
Therefore, the present study aims to evaluate the efficacy
of Hematoporphyrin coated Gold Nanoparticles on sur-
vival rate, egg-laying capacity of B. alexandrina snails
and on their hermaphrodite gland cells histologically.
2. MATERIALS AND METHODS
2.1. Snails
Biomphalaria alexandrina snails (6 - 9 mm) were
laboratory produced in Medical Malacology Department
at Theodor Bilharz Research Institute (TBRI), Giza,
Egypt.
2.2. Hematoporphyrin Coated Gold
Nanoparticles
A stock Mole/decimeter 3 Hpds concentration Mole/
decimeter 3 was prepared by dissolving 672 mg of Hpds
in 100 ml of glycerol by stirring overnight and kept in
dark. In 250 ml conical flask 5 ml of the stock solution
was added to 100 ml of glycerol and boiled with stirring.
To the solution 0.01699 g HAuCl4 was added and the
mixture continue boiling until the color of the mixture
changes from red to win red. Heating was removed and
stirring continued till cooling to room temperature. The
final cocentration is 5 × 104 Mdm3 (Hpd + Au).
2.3. Molluscicidal Activity of
Hematoporphyrin Coated Gold
Nanoparticles
Two concentrations of Hematoporphyrin coated Gold
Nanoparticles (5 × 10–6, 5 × 105 Mdm3) were prepared
according to El-Tarky (2005) and Ragheb (2009) results,
as 10 - 4 Mol/dm3 from each of Hpd and GNPs was
lethal to B. alexandrina and B. truncatus snails, respec-
tively. Therefore, B. alexandrina (6 - 8 mm) were incu-
bated for 12 hours at each tested concentration in the
dark, thereafter they were exposed to light irradiance
(336.2 W/m2) for either 2 or 4 hours. Three replicates for
each light exposure period, each of 10 snails/500 ml,
were prepared. After that, sn ails were washed thoroughly
with dechlorinated water and maintained in clean water
for 24 hours of recovery then mortality rates were re-
corded. Control snails were treated simultaneo usly as the
tested ones, without exposure to light irradiance. Death
of snails was determined by changes in appearance of
the shell and internal body (No lan et al., 1953).
2.4. Effect of Hematoporphyrine Coated
Gold Nanoparticles on Snail’s
Egg-Laying Capacity
B. alexandrina snails (7 - 9 mm) were incubated once
for 12 hours in the dark with sub lethal concentrations (5
× 105 and 5 × 106 Mdm3) of Hematoporphyrin coated
gold nanoparticles, after that they were exposed to direct
sunlight of 336.2 W/m2 for 4 hours. Then, they were
transferred to clean dechlorinated water and maintained
under laboratory conditions (25˚C) for 4 weeks. Two
replicates, each of 15 snails/L dechlorinated water, were
used for each concentration and control group. Each
aquarium was provided with polythylene sheets for ovi-
position. The snails were daily fed oven dried lttuce
leaves, dead ones were removed and the survival rate
were recorded weekly. The survivorship (Lx) and num-
ber of eggs/snails (Mx) were recorded weekly (South-
wood, 1978).
2.5. Histology
Histological preparations were done for snails incu-
bated for 12 hours to the two concentrations, 5 × 106
and 5 × 105 Mdm3, followed by 2 and 4 hours exposure
to direct sunlight (336.2 W/m2). Control snails were
treated with these concentrations without exposure to
light irradiance. The hermaphrodite gland of treated and
control snail groups was carefully incised using fine
K. El-Hommossany et al. / Open Journal of Animal Sciences 1 (2011) 54-60
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
56
scissors and dropped into a fixative Bouin’s solution,
then the sections (5 - 8 µm) were stained with delafield’s
haematoxylin and eosin according to Mohamed and
Saad (1990).
2.6. Statistical Analysis
Survival rates of treated snails were analyzed by
Chi-square values of contingency tables (Southwood,
1978).
3. RESULTS
From Table 1, it is clear that 5 × 105 Mdm3
(HpdGNPs) with 4 hours of exposure to 336.2 w/m2
sunlight suppressed the survival rate of B. alexandrina
snails by 50%. Meanwhile, con trol snails incubated with
5 × 10–5 Mdm–3 HpdGNPs were not affected and still
alive (100%). It is also, seen that snail’s survival rate
was concentration and light period dependent. So, in-
creasing the concentration from 5 × 106 to 5 × 105
Mdm3 reduced the survival rate from 100% to 62.7%,
respectively. Similar conclusion was observed by elon-
gation of the light exposure period from 2 to 4 hours, as
the rates of snails incubated with 5 × 10–5 Mdm–3 de-
creased from 62.7% to 50%, respectively.
The data in Ta ble 2 and (Figure 1 and 2) represent
the effect of 5 × 10–6 and 5 × 10–5 Mdm3 Hpd coated
GNPs on snail’s fecundity (Mx) and their survivorship
(Lx) post 4 weeks of recovery followed their exposure
once to this compound. It was observed that survivorship
(Lx) of treated snails was significantly decreased during
the 1st, 2nd and 3rd weeks of recovery in comparison with
that of control group (P < 0.01). The Lx values of snails
exposed to 5 × 106 and 5 × 105 Mdm3 Hpd coated
GNPs at the 1st week were 0.75 and 0.46, respectively
compared to 1.00 for control ones. It was also, noticed
that survived sn ails at 5 × 10–5 Mdm–3 Hpd coated GNPs
tried to somewhat overcome the harmful effects of the
tested compound through the 2nd and 3rd weeks of recov-
ery as their Lx was stable, being 0.46, but they suffered
from another significant reduction in this parameter at
the 4th week (Lx = 0.23) compared to 0.6 for control
ones (P < 0.05).
Ta ble 1 . Survival rate (%) of Biomphalaria alexandrina snails
exposed to Hematoporphyrin coated gold nanoparticles (12
hours incubation, and 4 hours exposure to 336.2 W/m2, 24
hours recovery).
Concentrationm Survival rate (%)
after Light exposur
for
(Mdm–3) 2 hours 4 hours
5 × 106 100 66.7
5 × 105 62.7 50.0
Control* 100 100
*Incubated with 5 × 105 Mdm 3 without light exposure.
For snail’s fecundity (Mx), treated snails laid low
number of eggs throughout the recovery period (4
weeks), in comparison with that of control ones. The
highest value of Mx for snails treated with 5 × 105
Mdm3 Hpd coated GNPs was recorded at the 3rd week
of recovery period, being 6.7 eggs/snail, compared to
37.6 eggs/contro l snail. Th is has a n egativ e reflect o n the
reproductive rate (Ro) of treated snails as it was 20.3 and
11.8 for groups exposed to 5 × 10–6 an 5 × 10–5 Mdm–3
of this compound, respectively, compared to 84.8 for
control group. The reduction rates of this parameter in
this case were 76.1% and 86.1%, respectively.
Histological effects on the hermaphrodite gland of B.
alexandrina snails revealed that the hermaphrodite gland
of control snails (Plate 1) consists of a number of
closely connected tubules where each tubule is lined
with distinct germinal epithelium gives rise to all the
stages of oogenesis and spermatogenesis. Oogonia are
found in groups which are arranged along the periphery
of the tubules. Oocytes are usually aggregated in the
lumen of the tubules. The mature ova deeply stained
with H-E than oocytes due to the accumulation of yolk
material in their cytoplasm. On the other hand, sper-
matogonia become arranged in small groups along the
side walls of the tubules. As a result of their division,
spermatocytes are produced. Spermatids are developed
from the secondary spermatocytes and are found in small
groups around them. Spermatozoa (sperms) are found in
large groups free in the lumen; each consists of an oval
head and thread like long tail (Mohamed and Saad,
1990).
For snails treated with Hpd coated GNPs (5 × 106
and 5 × 10–5 Mdm3), Plates 2 and 3 indicated that the
cells of their hermaphrodite gland suffered from injuries
in spermatocytes, oocytes, besides several degen erations
then evacuations were seen in many gonad’s cells. The
acini lost their normal shape and an almost damage to
their connective tissues was noticed which sev erely sup-
pressed their capacity for egg-laying .
4. DISCUSSION
The present lethal effect of the photosensitizer Hema-
toporphyrin coated gold Nanoparticles (Hpd coated
GNPs) against B. alexandrina snails was in parallel with
that of Hpd on L. natalensis (Abd El-Kader et al., 2001)
and B. alexandrina snails (El-Tarky, 2005 and El-Sayed
and El-Sherbini, 2006). As well, the harmful effects of
GNPs against B. truncatus snails ( Ragheb, 2009) su ppor t
the present study.
Concerning snail’s fecundity (Mx) and reproductive
rate (Ro), the present results showed that exposure of B.
alexandrina snails to Hpd coated GNPs markedly re-
duced these parameters. This could be attributed to the
K. El-Hommossany et al. / Open Journal of Animal Sciences 1 (2011) 54-60
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
5757
Table 2. Survivorship (Lx) and fecundity (Mx) of Biomphalaria alexandrina snails exposed to Hematoporphyrin coated gold
nanoparticles (12 hours incubation, 4 hours exposure to 336.2 W/m2 sunlight).
Control 5 × 10–6 Mdm–3 5 × 10–5 Mdm–3 Observation
Period (week) Lx M
x L
xMx L
x M
x L
xMx L
x M
x L
xMx
0 1.00 6.7 6.7 1.00 6.7 6.7 1.00 6.7 6.7
1 1.00 14.0 14.0 0.75*** 3.7 2.8 0.46*** 2.2 1.0
2 1.00 5.6 5.6 0.75*** 1.2 0.9 0.46*** 0.8 0.4
3 1.00 37.6 37.6 0.50*** 8.2 4.1 0.46*** 6.7 3.1
4 0.60 34.9 20.9 0.33n.s. 17.7 5.8 0.23* 2.6 0.6
Ro = 84.8 20.3 11.8
Reduction(%) 76.1 86.1
Lx = Survivorship, Mx = Mean number of eggs/snail/week; Ro = net reproductive rate, Sum LxMx; ***= Highly Significant, P < 0.01; *= Significant, P < 0.05; n.s.
= Not significant, P > 0.05.
0
0.2
0.4
0.6
0.8
1
1.2
01234
Observa ti on P eriod (W ee ks)
Survivorship (L
x
)
Figure 1. Survivorship (Lx) of Biomphalaria alexandrina snails exposed to Hematoporphyrin coated
gold nanoparticles (12 hrs incubation and 4 hrs exposure to 336.2 W/m2 sunlight).
-100
-80
-60
-40
-20
0
20
40
60
80
100
Re duction of R
o
R e pr oduction ra te (R
o
)
Ro % Reduction
Figure 2. Reproduction rate (Ro) of Biomphalaria alexandrina exposed to Hema-
toporphyrin coated gold nanoparticles (12 hrs incubation and 4 hrs exposure to
336.2 W/m2 sunlight).
Control
5 x 10-6 Mdm-3
5 x 10-5 Mdm-3
5 × 10–6 Mdm–3 5×10–5Mdm–3
Control
K. El-Hommossany et al. / Open Journal of Animal Sciences 1 (2011) 54-60
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
58
Plate 1. Light photomicrograph transverse section in the hermaphrodite gland of unex-
posed Biomphalaria alexandrina snails (Control), x 500. O=ova; Og = Oogonia; Oc=
oocyte; Sg = Spermatogonia; St = Spermatid; S= sperms.
Plate 2. Light photomicrograph transverse section in the Hermaphrodite gland of B. alexan-
drina snails treated with 5x10-6 Mdm-3 Hematoporphyrin coated Gold nanoparticles, ex-
posed to direct sunlight for 4hrs, x 500. D = Degenerated cells; V = Vacuoles.
harmful effects of this compound on physiological ac-
-tivities and reproductive system of treated snails, hence
considerably reduced their oviposition. This was sup-
ported by the present histological results (Plates 2 and 3)
on the hermaphrodite gland of snails exposed to this
compound. These plates revealed marked damages to the
gland cells in addition to several evacuations of its tu-
bules from gametogenic stages. This should greatly re
duce the offspring of survived snails, hence has a nega-
tive reflect on schistosomiaisis transmission. These ob-
servations are in accordance with the previous ones on
Hpd against oviposition of B. alexandrina snails (El-
Sayed and El-Sherbini, 2006) and on GNPs against re-
productive rate of B. truncatus snails (Ragheb, 2009).
Oc
O
S
O
O
g
St
S
g
V
D
K. El-Hommossany et al. / Open Journal of Animal Sciences 1 (2011) 54-60
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
5959
Plate 3. Light photomicrograph transverse section in the hermaphrodite gland of B .alexandrina
snails treated with 5 × 105 Mdm3 Hematoporphyrin coated Gold nanoparticles, exposed to direct
sunlight for 4hrs, x 500.D = Degenerated cells.
The harmful effects of Hpds could be partially due to
their hydrophobic character, hence localization in and
damage the cellular membrane leading to cells death
(Ben Amor et al, 1998). As well, these photosensitizers
damage the biological targets by photosensitized oxida-
tion which deactivate certain enzymes through destruc-
tion of specific amino acids (e.g. Methionine and tryp-
tophan), nucleic acid (Primarily of guanine) and by oxi-
dation of unsaturated fatty acids and cholesterol in cell
membrane (Abd El-Meguid, 1996). In addition, precipi-
tated GNPs inside snail’s tissues when irradiated with
some forms of light energy they absorb light and emit
localized heat with its lethal or traumal effects (Jana et al,
2007).
It is concluded from the present work that exposing B.
alexandrina snails to sublethal concentrations of the
photosensiter Hpd coated GNPs (12 hours incubation, 4
hours exposure to 336.2 W/m2) significantly reduced
their reproductive capacity that may have a negative
reflect on schistosomiaisis transmission.
REFERENCES
[1] Abd El-Kader, M.H., El-Sherbini, S.A., El-Tayeb, T.A.,
Mohamed, L.F. and Mohamed, S.A. (2001) Hematopor-
phyrin as photomolluscicide to control Fasciola snails.
Proceedings of the 4th International Europe Mediterra-
nean Conference on Laser, Photobiol og y, Applications in
Medicine, Cairo University, Cairo, 13-16 February 2001,
46.
[2] Abd El-Meguid (1996) Effect of He-Ne laser beam on
the Ommatidia in Schistsocerca gregaria. Arab Journal
of Nuclear Sciences and Applications, 29, 53-67.
[3] Ben Amor, T., Tranchin, M., Bortolotto, L., Verdiglione,
R. and Jori, G. (1998) Porphyrin and related compounds
as photoactivable Insecticides. Photochemistry and
Photobiology, 67, 206-211.
doi:10.1562/0031-8655(1998)067<0206:PARCAP>2.3.C
O;2
[4] Bertoloni, G., Sacchetto, R., Jori, G., Vernon, D.I. and
Brown, S.B. (1993) Portophyrin photosensitization of
Enterococcus hirae and Candida albicons cells. Lasers in
the Life Sciences, 5, 267-275.
[5] El-Sayed, K.A. and El-Sherbini, S.A. (2006) Impact of
Hematoporphyrin and different laser sources on Biom-
phalaria alexandrina snails and their infection with
Schistosoma mansoni. Journal of Biological Chemistry,
Environmental Sciense, 1, 319-340.
[6] El-Tarky, A.G.S. (2005) Semifield studies to control
schistosomiaisis free larval stages and its snail vectors
using selected sensitizers with sunlight and laser radia-
tion. Ph.D. Thethis, National Institute of Laser Enhanced
Sciences, Cairo University, Cairo.
[7] Jana, N.R., Earhart, C. and Ying, J.Y. (2007) Synthesis
and covalent functionalization of nonoxidic iron core
shellnanomagnetic. Chemistry of Materials, 19, 5074.
doi:10.1021/cm071368z
[8] Lardans, V. and Dissous, C. (1998) Snail control strate-
gies for reduction of schistosomiasis transmission. Para-
sitology To day, 14, 413-417.
doi:10.1016/S0169-4758(98)01320-9
[9] Mohamed, S.H. and Saad, A.A. (1990) Histological stud-
ies on the Hermaphrodite gland of Limnaea caillaudi and
Biomphalaria alexandrina upon infection with certain
larval trematodes. Egyptian Journal of Histology, 13,
D
K. El-Hommossany et al. / Open Journal of Animal Sciences 1 (2011) 54-60
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
60
47-53.
[10] Nolan, M.O., Howard, W.B. and Elizabeth, R.M. (1953)
Results of laboratory screening tests of chemical com-
pounds for molluscicidal activity. The American Journal
of Tropical Medicine and Hygiene, 22, 716-752.
[11] Pasvol, G. and Hoffman, S. (2001) Trop. Med. Science
and Practice “Schistosomiasis”. Imperial College Press, 3,
12-17.
[12] Ragheb, M. (2009) Histological studies on the effect of
gold nanoparticles on Scistosoma haematobium interme-
diate host (Bulinus truncates snails). MSc. Thesis, Zool-
ogy Departement, Fuculty of Science, Cairo University,
Cairo.
[13] Southwood, T.R.E. (1978) Ecological methods, with
particular reference to the study of insect populations.
The English Languag e Book Society and Chopan-Hall.
[14] Spikes, J.D. (1984) Photobiochemistry of porphyrins. In:
Dorion D.R. and Comer, C.Z. Eds., Porphorin Localiza-
tion and Treatment of Tumors, Alan R. Liss, New York,
pp. 19-39.