الفهرس 
Water temperatureOnly 14 pages are availabe for public view
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Abstract
SUMMARY
Along a year from March 2012 to January 2013, the present study
was carried out to determine the environmental conditions which affect
the distribution and density of some freshwater snails in certain water
canals representing four Egyptian governorates (El-Giza, El-Ismailia, El-
Gharbeya and El-Menoufiya). Also, the correlation between such
conditions and the abundance and percentages of terrestrial fungi living
in the surveyed water canals and adjacent soil was recorded. In addition,
the study aimed at finding out a new molluscicidal agent from a
biological origin instead of chemical molluscicides which adversely
affect the environment. In this respect screening tests were carried out on
236 fungal isolates, 20 species were effective against B. alexandrina
snails causing 100% death of snails at 10% concentration of the fungal
filtrate.
The LC50 values were calculated, where the most effective species
were Penicillium janthinellum and Aspergillus terreus. The sublethal
concentrations of these two species were used for further tests. Both
fungal filtrates decreased survival rate of treated snails, LC5 and LC25 of
Aspergillus terreus and Penicillium janthinellum, respectively reduced
egg-laying capacity and reproduction of B. alexandrina snails.
Deleterious effects on both hermaphrodite and digestive glands of snails
exposed to sublethal concentrations of the 2 species were observed.
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155
Hemolymph proteins of treated snails were examined using SDSPAGE.
There were many bands disappeared in the treated snails and
present in the control group and vice versa. The filtrate of Penicillium
janthinellum, which was the most effective amongst all fungi tested,
underwent chemical separation and analysis to determine the
molluscicidal fraction. It was identified as methyl gallate. Sublethal
concentrations of this purified identified compound was also tested on
hemolymph proteins to make a comparison with its filtrate counterpart.
Finally, the toxicities of sublethal concentrations of both filtrates, besides
the effective fraction (methyl gallate) were investigated on Daphnia
pulex which is a zooplankton used as a biological indicator of water
pollution. This final step was performed to ensure the safety of
application of the fungal sublethal concentrations.
The present results showed the following:
- The minimum mean of temperature recorded was that of surveyed water
canals in El-Ismailia governorate (16.05°C) during winter, while the
maximum mean was that observed in El-Menoufiya governorate
(30.75°C) in both spring and summer.
-The minimum and maximum mean pH values were recorded during
summer and winter at El-Ismailia governorate (6.85 and 8.8 respectively)
- The lowest mean value of electrical conductivity (EC) was detected in
the spring at El-Giza governorate (1.72 μS/cm), while the highest value
was observed in autumn at El-Gharbeya governorate (804.5 μS/cm).
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156
-The minimum and maximum mean values of total dissolved solids were
observed in El-Ismailia governorate during spring and summer,
respectively (13.6 and 1592 ppm).
-The least mean value of dissolved oxygen was recorded in winter of El-
Menoufiya governorate (0.21 ppm), while the highest value was recorded
in El-Gharbeya governorate during summer (10.15 ppm).
- A total of 1182 snails represented by 11 species belonging to class
Gastropoda were collected from the sites of investigation in the four
surveyed governorates during the study period. These species were
Biomphalaria alexandrina, Bulinus truncatus, Lymnaea natalensis,
Planorbis planorbis, Physa acuta, Helisoma duryi, Melanoides
tuberculata, Cleopatra bulimoides, Bellamya unicolor, Lanistes
carinatus and Succinia cleopatra.
-The highest percentages of Biomphalaria alexandrina snails was
recorded during winter (94.7%), spring (96.7%), spring (76.4%) and
autumn (100%) in El-Giza, El-Ismailia, El-Gharbeya and El-Menoufiya
governorates, respectively. Also, the abundance and percentages of other
snail species differ from season to another in the surveyed governorates.
- Spring was the richest season with different snail species in El-Giza and
El-Gharbeya governorates. While, the variation in snail species collected
from El-Ismailia showed its maximum in both spring and winter. In El-
Menoufiya governorate, the richest season with snails was winter.
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157
- A total of 2946 fungal isolates belonging to 8 main genera of fungi:
Aspergillus, Penicillium, Fusarium, Rhizopus, Alternaria, Trichoderma,
Acremonium and Microascus were isolated from the sites of
investigation in the four surveyed governorates during the study period.
-Seasonal variations of fungal species in El-Giza, El-Ismailia and El-
Menoufiya governorates showed that Aspergillus spp. reached its
maximum percentage during autumn. While in El-Gharbeya governorate,
the highest Aspergillus percentage was observed in summer. The highest
percentages of Penicillium spp. were recorded twice during summer in
both El-Ismailia and El-Menoufiya governorates. Fusarium spp. were
highly represented during spring in each of El-Giza, El-Ismailia and El-
Gharbeya governorates.
Acremonium spp. appeared only in summer in both El-Ismailia and
El-Gharbeya governorates, while completely disappeared from El-
Menoufiya governorate. Microascus sp. was isolated only from El-
Gharbeya governorate during spring. Rhizopus spp. exhibited their
maximum percentages in the four surveyed governorates during winter.
- The maximum variation of fungal groups was observed during spring in
each of El-Ismailia, El-Gharbeya and El-Menoufiya governorates. On
contrary, seven from the eight studied genera were represented in autumn
of El-Giza governorate.
-The highest percentages of aquatic plants abundance were those of
Lemna gibba and grasses (75% for both), followed by Azolla pinnata
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(50%) in El-Giza governorate. On the other hand, Eichhornia crassipes
was the most abundant aquatic plant in El-Ismailia governorate as it was
represented by 100% followed by Atropa belladonna. Also, Eichhornia
crassipes was the most abundant in El-Gharbeya governorate with a
percentage equal 62.5%. Concerning El-Menoufiya governorate, the
highest percentage of abundance amongst aquatic plants was that of
Eichhornia crassipes (100%) followed by both Lemna gibba and grasses
which recorded the same percentage (87.5%).
- Correlation between physico-chemical parameters and snails collected
in the present study revealed that temperature was negatively correlated
with B. alexandrina, Physa acuta and Succinia cleopatra. On the other
hand, pH was positively correlated with Lymnaea natalensis, while both
negative and positive correlations were recorded between pH and B.
alexandrina in El-Menoufiya and El-Giza governorates, respectively.
Electrical conductivity (EC) was negatively correlated with both
Bulinus truncatus and Helisoma duryi. Moreover, a significant negative
correlation was observed between total dissolved solids on one hand and
each of B. alexandrina, Bulinus truncatus and Helisoma duryi on the
other hand. A positive correlation between TDS and each of Cleopatra
and Succinia was observed. Furthermore, Dissolved oxygen was
positively correlated with each of B. alexandrina, Bulinus truncatus,
Cleopatra bulimoides, Lanistes carinatus and Melanoides tuberculata
snails.
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- The relationship between physico-chemical parameters and fungi
showed a strong positive correlation between temperature and
Aspergillus. Although pH was negatively correlated with Alternaria spp.,
it was positively correlated with Rhizopus spp. Other significant positive
correlations were found between TDS and each of Trichoderma spp. and
Rhizopus spp. Moreover, electrical conductivity was positively correlated
with Penicillium spp., concerning the relations between dissolved oxygen
and each of Trichoderma spp. and Acremonium spp., they were either
positive or negative according to the governorate from which samples
were collected. Penicillium spp. were positively correlated with dissolved
oxygen.
-Correlations between aquatic plants and snails declared that B.
alexandrina has positive correlations with each of Lemna gibba and
Equisetum arvens, while negatively correlated with Atropa belladonna.
In addition, Bulinus truncatus and Melanoides tuberculata were
positively correlated with Eichhnoria crassipes, while negatively
correlated with grasses and Pistia stratiotes. Concerning Physa acuta and
Lymnaea natalensis, the former snail showed negative correlations with
both Lemna gibba and Ceratophillam demersum, while the second one
had negative relation with Equisetum arvens. Furthermore, Helisoma
duryi was positively correlated with L. gibba while negatively correlated
with E. crassipes and A. belladonna. Positive correlation was observed
between Planorbis planorbis and Equisetum arvense, while negative
correlation between the same snail and Atropa belladonna was recorded.
Cleopatra and Lymnaea showed positive correlations with A. belladonna.
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- Correlations between isolated fungi and collected snail species were
determined. Although some positive relations were observed, the great
majority were negative, like the correlations between Bulinus truncatus
and each of Aspergillus and Alternaria spp. Moreover, Lymnaea
natalensis and each of Alternaria and Rhizopus spp. were negatively
correlated, also, Fusarium and each of Succinia cleopatra and Physa
acuta exhibited negative correlations. Finally, the relation between
Melanoides tuberculata and Aspergillus was negative. Furthermore,
certain seasons which were the richest with snails, were in the same time
the poorest with fungi; this was the case in the spring of El-Giza
governorate and the winter of both El-Ismailia and El-Menoufiya
governorates.
- Amongst 236 fungal isolates, 20 fungal species were molluscicidally
active against adult B. alexandrina snails. They result in 100% death of
the tested snails at a concentration of 10%. They were 11 Aspergillus
spp. (Aspergillus niger var. awamori, A. terreus, A.terreus var. africanus,
A. clavatus, A. flavus, A. petrakii, A. niveus, A. viridinutans, A. tamarii,
A. oryzae and A. parasiticus), 3 Penicillium spp. (Penicillium
implicatum, Penicillium janthinellum and Penicillium citrinum), 2
Acremonium spp. (A. alabamense and A. spinosum), Fusarium
proliferatum, Trichoderma koningii, Rhizopus azygosporus and
Microascus manginii. from the 20 effective fungal filtrates, the
minimum LC50 values were those of Penicillium janthinellum (1.03%)
and Aspergillus terreus (1.05%)
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-Sublethal concentrations of the two effective fungal species were
calculated: LC5= 0.1%, LC15=0.3% and LC25= 0.5%
-The survival rates of snails treated with sublethal concentrations of
Aspergillus terreus were investigated through 20 successive weeks. It
was found that after 4 weeks of exposure to LC5 of A. terreus, the
survival rate of snails decreased to 55%, while that of snails treated with
LC15 and LC25 reached 50% as compared with 100% for control snails.
At the 11th week, the survival rate of the snails treated with LC15
decreased to 15% in comparison with 95% for control group. These
values continued to decrease as the time extended, by the 19th week, only
10% of each tested group of snails survived as compared with 90% in the
control group.
-The survival rate of snails exposed to LC25 of P. janthinellum decreased
sharply to 45%, while that of control was 100% at the 4th week of
exposure. Moreover, a gradual reduction of this rate was recorded, thus
its values were 20% for both groups of snails treated with LC5 and LC15,
while reached 0.05% for the snails treated with LC25 at the 7th week of
exposure, the survival rate of control was 95%. It was also found that
prolonged exposure (15 weeks) resulted in the death of all snails exposed
to LC25, while 90, 15 and 10 % of snails survived in control, LC5 and
LC15 groups, respectively. By the 20th week, all the snails in LC15
treatment died, while the survival rate of control group was 85%.
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- The mean eggs/snail/week for snails treated with LC5, LC15 and LC25 of
A. terreus were 5.48, 7.05 and 6.05 respectively, while for P.
janthinellum, these values were 3.09, 4.95 and 5.16 as compared with
10.79 eggs for control snails. Featuring the variation in reproduction
along the five months of exposure declared that, in case of snails treated
with sublethal concentrations of A. terreus, it was found that during the
first month, there was a positive relationship between sublethal
concentrations and reproduction. As the concentration increased from
LC5, LC15 till LC25, the reproduction increased from 5.94, 7.44 to 14.48,
respectively while that of control group was the highest (22). During the
second month, the values of reproduction for snails maintained at LC5
and LC15 were so close (10.2 and 9.26 respectively), while reproduction
of snails treated with LC25 continued to be higher than both mentioned
concentrations as it was 16.29. By the third month of exposure, the
reproduction noticeably decreased to 1.75 in snails treated with LC25 as
compared with 43.09, 4.5 and 5.2 for control, LC5 and LC15 groups,
respectively. At the 4th and 5th months, the least reproduction values were
observed in snail groups maintained at LC25 (2.25 and 2.1 respectively).
In case of snails treated with P. janthinellum, it was observed that
along the five months of exposure to the three tested sublethal
concentrations, the snails in the control group recorded the highest
reproduction value. During the first month, the maximum reproduction
appeared in the group of snails maintained in LC5, followed by LC25. In
the second month, there was a gradual decrease in the reproduction value
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as the concentration increased; 6.05, 5.1 and 1.49 for LC5, LC15 and LC25
groups, respectively. Moreover, the reproduction decreased as the
exposure time extended to three months where its value reached 1.2 for
snails treated with LC25. At the 4th month, the values of reproduction were
so close in the three tested concentrations with slight increase in LC15
group. This increase continued in the 5th week reaching 3.35 as compared
with 1.94 and zero for snails maintained in LC5 and LC25, respectively.
-The percentages of hatchability of B. alexandrina eggs exposed to
sublethal concentrations of A. terreus were 81.82 and 82.5% for one day
old eggs exposed to LC5 and LC15, respectively compared with 96.25 %
for control eggs, while this percentage reached 78 for eggs exposed to
LC25.On the other hand, the percentages of hatchability for three days old
eggs were decreased as the concentration increased; it was 68.75, 55 and
42.7% for eggs exposed to LC5, LC15 and LC25, respectively compared
with 94.7% for control eggs.
-Concerning eggs exposed to sublethal concentrations of P.
janthinellum, it was revealed that as the concentration increased, the
hatchability percentages decreased. The minimum percentages were
those of eggs exposed to LC25, as they were 71.8 and 43.21% for one and
three days old eggs, respectively. In general, it was observed that the
hatchability percentages of three days old eggs were less than those of
one day old eggs exposed to sublethal concentrations of both effective
fungi. It was clear that all the tested sublethal concentrations from both
fungal filtrates adversely affect hatchability percentages of eggs (P<0.05).
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-Histological studies on hermaphrodite gland of treated snails showed
that the snails treated with LC5 of A. terreus were characterized by
degeneration and deformation of mature ova, also early stages of
oogenesis were not represented.
The effect of LC15 of A. terreus was weaker than that of LC5, there
was degeneration of mature ova and primary spermatocytes were densely
stained.
Concerning the effect of LC25, it was observed that the acini were
more or less similar to that of control as all stages of oogenesis and
spermatogenesis were represented, but the connective tissue between
acini was degenerated.
-For snails treated with LC5 of P. janthinellum, the degeneration of some
ova and the deformation of others were the most obvious effects. As the
concentration increased to LC15, more adverse effects were noticed as
shrinkage and disruption of acini took place, besides the degeneration
and deformation of mature ova.
In case of snails’ exposure to LC25of P. janthinellum, acini appeared
almost vacant, only few mature ova and primary spermatocytes were
represented in certain acini.
-Histlogical studies on digestive gland declared that, for snails treated
with LC5 of Aspergillus terreus, vacuolation of digestive cells and
degeneration of secretory ones were observed. Moreover, some secretory
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cells were densely stained, and few secretions appeared in the digestive
cells as the concentration increased to LC15.At LC25, clear necrotic
changes of the digestive tubule and erosion of the cytoplasm of digestive
cells were observed. Moreover, the tubular membrane was ruptured.
Similarly, the snails treated with LC5 of P. janthinellum showed
vacuolation of digestive cells and degeneration of secretory ones. As the
concentration increased to LC15, the effect was more pronounced; the
cytoplasmic cell boundaries of digestive cells were not clearly
distinguished and lumen nearly disappeared. Exposure of snails to LC25
resulted in hydropic degeneration as clear necrotic changes of the
digestive tubule and erosion of cytoplasm of digestive cells occurred,
besides noticeable degeneration of secretory cells.
- Chemical separation of Penicillium janthinellum filtrate components
was carried out, where 36 fractions were separated, these fractions were
tested on Biomphalaria alexandrina snails to determine the effective one.
The effective fraction underwent a series of chemical analyses to identify
its functional groups and chemical structure. The results of such analyses
were explained and the compound was identified as methyl gallate.
-The electrophoretic separation of hemolymph proteins from B.
alexandrina showed different effects of the tested sublethal
concentrations. Treatment of snails with LC5 of A. terreus yielded a
complex pattern of polypeptides ranged in molecular weight from 16.06
to 177.75 KDa, this range increased to 190.78 KDa as the concentration
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increased to LC15. It was from 14.93 to 195.34 KDa in case of snails
exposed to LC25. The minimum number of bands (14) was observed in
the group treated with LC5 compared with 18 bands in the control group,
also the minimum value of similarity index was that of polypeptides
separated from snails exposed to LC5. There were many bands
disappeared in the treated snails and present in the control group and vice
versa. The band which has a molecular weight equals 108.2 KDa
disappeared in the snails treated with LC5, while the 71.911 and 30.171
KDa bands disappeared in both groups treated with LC5 and LC25. The
bands 177.75 and 45 KDa disappeared in the snails exposed to LC15 and
LC25. On the other hand, there were 10 excess bands of proteins
separated from snails exposed to LC15, while 9 and 8 excess bands were
observed in snails treated with LC5 and LC25, respectively.
-The protein bands separated from snails treated with LC5, LC15 and LC25
of Penicillium janthinellum were 14, 13 and 14 bands, respectively
compared with 18 bands from the control snails. The bands with
molecular weights equal 116.75, 45 and 40.455 KDa disappeared in both
groups of snails exposed to LC15 and LC25. While the 61.142 KDa band
disappeared in both LC5 and LC15 treated snails. There were 7, 8 and 9
excess bands in snails treated with LC5, LC15 and LC25, respectively. The
minimum similarity index was that of protein bands separated from snails
exposed to LC25.
-The number of protein bands separated from snails exposed to LC5,
LC15 and LC25 of the identified effective compound from P. janthinellum
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(methyl gallate) were 12, 13 and 15 bands, respectively compared with
15 bands from control group. The 98.979 and 69.954 KDa bands were
shared between LC15 and LC25 treated snails. While 15.987 KDa band
was shared between LC5 and LC15 groups. The highest number of shared
bands was recorded between control and LC25 treated snails (similarity
index=0.8). While the minimum similarity index with control group was
that of snails treated with LC5 of methyl gallate as it equals 0.22.
-Cladocerans are ecologically very important members of freshwater
invertebrates and amongst them Daphnia pulex has been often utilized as
test organisms for the ecotoxicological monitoring of aquatic ecosystems.
These organisms were used in the present study to evaluate the safety of
application of both fungal filtrates, besides methyl gallate as a pure
compound. It was observed that 10% and 20% of Daphnia pulex
individuals died after their exposure to LC15 and LC25 of A. terreus
filtrate, while exposure of these organisms to LC25 of P. janthinellum
filtrate resulted in only 10% death, no mortality was observed in both
LC5 group and control organisms.
Concerning the identified compound, it was found that 3.3% of the
tested D. pulex died in LC25, while there was no mortality in individuals
exposed to LC5 and LC15 groups