Separation of a compound effective against Biomphalaria alexandrina snails from the filtrate of Penicillium janthinellum

Abstract

Biomphalaria alexandrina snails, as intermediate hosts of schistosomiasis, play a central role in dissemination of the disease. Control of these snails by chemical molluscicides adversely affects the aquatic environment, causing toxic and carcinogenic effects on non-target organisms. Searching for promising substances from biological origin becomes an urgent need to overcome these drawbacks. Screening tests were carried out on 236 fungal genera isolated from the habitat of freshwater snails in four Egyptian governorates. Twenty species were effective against B. alexandrina snails, but the most potent was Penicillium janthinellum as the value of LC50 was 1.03%. Chemical analyses of this filtrate resulted in the separation of a compound effective against snails; it was identified as methyl gallate. Protein electrophoresis showed that both fungal filtrate and methyl gallate affect the protein pattern of snails’ haemolymph. Little or no mortality of Daphnia pulex individuals was observed after their exposure to sub lethal concentrations of each treatment.

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Separation of a compound effective against
Biomphalaria alexandrina snails from the filtrate
of Penicillium janthinellum
Abd El-Halim A. Saad, Magdy T. Khalil, Fawzy M.A. Ragab, Amal A.I. Mekawey
& Marwa T.A. Abdel-Wareth
To cite this article: Abd El-Halim A. Saad, Magdy T. Khalil, Fawzy M.A. Ragab, Amal A.I. Mekawey
& Marwa T.A. Abdel-Wareth (2015): Separation of a compound effective against Biomphalaria
alexandrina snails from the filtrate of Penicillium janthinellum, International Journal of
Environmental Studies, DOI: 10.1080/00207233.2015.1082246
To link to this article: http://dx.doi.org/10.1080/00207233.2015.1082246
Published online: 17 Sep 2015.
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Separation of a compound effective against
Biomphalaria alexandrina snails from the filtrate
of Penicillium janthinellum
ABD EL-HALIM A. SAAD†, MAGDY T. KHALIL†, FAWZY M.A. RAGAB‡,
AMAL A.I. MEKAWEY§AND MARWA T.A. ABDEL-WARETH*‡
†Faculty of Science, Department of Zoology, Ain Shams University, Abbasia, 11566 Cairo, Egypt;
‡Department of Environmental Research and Medical Malacology, Theodor Bilharz Research
Institute, Imbaba, 12411 Giza, Egypt; §The Regional Centre for Mycology and Biotechnology,
Al-Azhar University, Nasr City, 11754 Cairo, Egypt
Biomphalaria alexandrina snails, as intermediate hosts of schistosomiasis, play a central role in
dissemination of the disease. Control of these snails by chemical molluscicides adversely affects
the aquatic environment, causing toxic and carcinogenic effects on non-target organisms. Searching
for promising substances from biological origin becomes an urgent need to overcome these draw-
backs. Screening tests were carried out on 236 fungal genera isolated from the habitat of freshwater
snails in four Egyptian governorates. Twenty species were effective against B. alexandrina snails,
but the most potent was Penicillium janthinellum as the value of LC
50
was 1.03%. Chemical analy-
ses of this filtrate resulted in the separation of a compound effective against snails; it was identified
as methyl gallate. Protein electrophoresis showed that both fungal filtrate and methyl gallate affect
the protein pattern of snails’haemolymph. Little or no mortality of Daphnia pulex individuals was
observed after their exposure to sub lethal concentrations of each treatment.
Keywords:Penicillium janthinellum;Biomphalaria alexandrina; Protein electrophoresis; Methyl
gallate
Introduction
Among human diseases caused by parasites, schistosomiasis is second to malaria in its
socio-economic and public health impact in tropical and subtropical regions of the world.
Schistosomiasis is endemic in 76 tropical developing countries, and 600 million people are
at risk of acquiring the disease. There are estimates that up to 200 million are already
infected. Extreme poverty and poor sanitary conditions are major risk factors for the
disease, along with inadequate public health infrastructure [1].
Two of the five most important human species of Schistosoma are endemic in Egypt;
S. haematobium, which mainly causes disease in the urinary tract, and S. mansoni, which
mainly causes morbidity in the gut and liver. The intermediate hosts for S. mansoni are
Biomphalaria alexandrina and Biomphalaria glabrata snails, while Bulinus truncatus
serves as intermediate host for S. haematobium.B. alexandrina snails are the subject of
many studies because they are widely distributed along the Nile Delta [2].
*Corresponding author. Email: marwatamim2001@hotmail.com
© 2015 Taylor & Francis
International Journal of Environmental Studies, 2015
http://dx.doi.org/10.1080/00207233.2015.1082246
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Snail control is a rapid and efficient method of reducing or eliminating transmission.
Application of chemical molluscicides causes biocidal action on non-target organisms,
besides causing genotoxicity and carcinogenic effects [3]. Accordingly, there has been
much recent work on natural enemies such as predators, parasites, and pathogens. There is
a need for a biological agent, possessing the desirable properties of a chemical mollusci-
cide making it highly toxic to the target organism, producible on an industrial scale, hav-
ing a long shelf life and being safely transportable [4]. Many studies have focused on
biological control methods such as the use of certain algae [5], bacterial strains [6] and
fungal extracts [7].
Fungi have been recovered from diverse aquatic habitats including rivers, streams, mar-
ine environments and aquatic sediments [8,9]. The fungi encountered in freshwater are
divided into two principal groups; the hydro fungi which require the presence of water to
complete their life cycle and geo-fungi or typical soil fungi which are not adapted to aqua-
tic existence. They might be found in water because of adequate supply of nutrients, and
have been regarded as ‘facultative aquatic fungi’[10]. Filamentous fungi represent an
important source of natural products [11].
Materials and methods
Materials
Growth media and test species
Sabouraud agar (SA) and Czapek agar (CZA) media were used to isolate and identify
fungi from both water and soil samples. Potato dextrose broth medium was used for grow-
ing fungal isolates to be tested as filtrates on B. alexandrina snails [12]. Daphnia pulex is
a zooplankton organism which is considered as a biological indicator of the environmental
hazard caused by the application of molluscicides [3]. D. pulex was collected from natural
snails’habitat and transferred to laboratory. They were maintained under laboratory condi-
tions, reared in de-chlorinated water in glass aquaria and fed on yeast [13].
Chemicals
Silica gel (G 100), chloroform and methanol were the chemicals needed to purify the
effective fraction from the crude fungal extract by column and thin layer chromatography
(TLC). Toluene–ethyl acetate–90% formic acid (TEF), as a solvent, and ceric sulphate as a
spray were used in TLC [14]. To investigate the alteration of protein pattern in treated
snails, sodium dodecyl sulphate and polyacrylamide gel were used to carry out protein
electrophoresis [15].
Methods
Study area
Samples of water and soil were collected from eight sites through a year from 2012 to
2013. These sites represent four Egyptian governorates as follows: El-Giza Governorate,
El-Ismailia Governorate, El-Gharbeya Governorate and El-Menoufiya Governorate.
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Samples collection
Water samples were collected in sterilized plastic containers, 20 cm below the water’s sur-
face [16]. The container was completely filled with water, and then the cap was replaced
immediately. They were labelled, kept in an ice box and then transported to the laboratory
for analysis within 12 h. Soil samples were collected from the banks of the investigated
canals using a surface sterilized trowel, and transported to the laboratory in labelled
polyethylene bags [17].
Isolation of fungi
From water samples: One millilitre of each water sample was spread, in triplicate, into
Petri dishes containing SA to which 500 mg/l of chloramphenicol was added, and then the
cultured plates were incubated at 28 °C (±2 °C) for 1 week [18].
From soil samples: 25 g of each soil sample were transferred to 250 ml of sterilized
water in 500 ml Erlenmeyer flasks. These flasks were then shaken at constant speed
(150 rpm) for 15 min at room temperature [19]. Then, they were left until complete sedi-
mentation of the soil had taken place. Serial decimal dilutions were made from the original
concentration. Then, 0.5 ml volumes were pipetted onto SA media. Three plates were
prepared for each concentration and were incubated at 28 °C for 1 week.
Identification and maintenance of fungi
The morphology of the fungal colonies was studied on Sabouraud dextrose agar and CZA
[20]. Universal manuals were used in the identification of fungal species [21,22]. These
species were maintained by continuous sub culturing on Sabouraud dextrose agar at
constant intervals, and the slants then kept refrigerated.
Screening and toxicity tests
Fungal cultures were prepared by inoculating conical flasks (250 ml capacity) containing
50 ml of potato dextrose broth medium with fungal discs (5 mm diameter) which were cut
from 7 days old cultures [12]. The inoculated flasks were incubated on a rotary shaker
(150 rpm) at 28 °C for 10 days. The mycelia then were separated by filtration, using a
membrane filter [23]. Different fungal filtrates were used in toxicity tests. A series of con-
centrations was prepared using de-chlorinated tap water at 22 ± 2 °C to determine the most
potent fungal species. Three replicates were used; each of ten snails (8–10 mm in
diameter). The exposure period was 24 h at room temperature. Another group of snails
was maintained under the same experimental conditions as a control group [24]. At the
end of the exposure period, these snails were removed from each tested concentration,
washed thoroughly with de-chlorinated tap water and transferred to another container for a
recovery period for 24 h. Then, dead snails were counted, and LC
50
and LC
90
values of
the most toxic fungal filtrates were computed [25].
Identification assay for the most effective compounds
Purification techniques were carried out to identify the effective molluscicidal compound
extracted from its fungal filtrate. The effective crude extract was subjected to fractionation
as follows:
Separation of a compound effective against B. alexandrina 3
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Column chromatography: Ten millilitres from the crude extract were chromatographed
on a column (1.5 cm diameter and 50 cm long) of silica gel (G 100) after activation at 80
(±2)°C for 30 min, then subjected to elution with chloroform and methanol (90:10) v/v. In
order to stabilise and equilibrate the bed, the gradient volume of crude extract was passed
through the column; the fractions (each of 1 ml) were collected separately [26,27]. Then
each of them was re-tested on the snails to determine the most effective one.
Thin layer chromatography: TLC plates (20 × 20 cm Merk aluminum sheet, silica gel
60, layer thickness 0.2 mm) were used. The diluted effective fraction was spotted at the
start of the silica gel plates, and allowed to dry before applying other spots. A spot of ter-
binafine dissolved in chloroform/methanol (3:1 v/v) was used as a reference standard.
Samples were chromatographed for 17 cm in TEF (5:4:1 v/v/v) in a solvent saturated
atmosphere, then allowed to air dry.
TLC plates were examined under white and UV light (365 nm) and the characteristics
of the spots were recorded. They were also examined under UV light (254 nm), and then
back to 365 nm to visualise the intensity of spots and calculate their rate of flow (R
f
) val-
ues. TLC plates were sprayed with ceric sulphate in 3 M sulphuric acid, and examined
under white and UV lamps (365 and 254 nm) [14,28].
Chemical analysis by spectral measurements
Infrared spectra (IR): The infrared absorption spectrum of isolated effective fraction was
estimated using an anicum infinity series FTIR, Perkin –Elmer 1650 Spectrophotometer™.
Nuclear magnetic resonance (NMR): The proton (
1
H) NMR spectra were estimated
using FT-NMR Bruker Ac 200 spectrometer™.
SDS–PAGE
Haemolymph sampling: Haemolymph was collected from the tested snails at the 4th week
post exposure. In each specified group, the haemolymph of 8–10 individual snails was
pooled in 1 ml Eppendorf tube. All haemolymph samples from each experimental group
were centrifuged at 5000 rpm for 5 min at 4 °C to pellet haemolymph and other particulate
materials [29]. The pellet was discarded and cell-free haemolymph was mixed with sample
buffer in a ratio of 4 part of haemolymph: 1 part sample buffer. Samples were boiled for
5 min at 100 °C in a water bath.
Electrophoretic analysis of haemolymph proteins: The protein profiles were analysed by
sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) [15]. The
similarity of the polypeptide profile between the different groups was obtained from Dice
similarity coefficient [30]:
S¼2a=2aþbþc
where (S) is the degree of identity, (a) is the number of common shared bands in two com-
pared samples, (b) is the number of excess bands in the first compared sample, and (c)is
the number of excess bands in the second compared sample.
An ‘S’value of 1.0 denotes complete identity in the electrophoretic profile of both
groups, while a value of <1.0 indicates a variation in the polypeptide profile between the
two compared samples.
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Toxicity tests on D. pulex
Healthy D. pulex (30 in each treatment) were exposed to sublethal concentrations (LC
5
,
LC
15
and LC
25
) of the effective fungal filtrate and the identified effective compound in
petri-dishes for 24 h, followed by 24 h as a recovery period. Another group was main-
tained in de-chlorinated tap water as control. Their viability was observed and mortality
percentages were calculated [31].
Results
Toxicity tests on B. alexandrina snails
Screening tests were carried out on 236 fungal isolates to determine the most effective
ones against adult B. alexandrina snails. It was found that 20 fungal species have mol-
luscicidal activity as they result in 100% death of the tested snails at a concentration of
10% (v/v) (table 1). Amongst them, the minimum LC
50
value was that of P. janthinellum
(1.03%) (table 2).
Identification of the effective compound from P. janthinellum
After column chromatography and TLC analyses; 36 fractions (each of 1 ml) from chloro-
form/ methanol extract of P. janthinellum were collected. These fractions were re-tested on
B. alexandrina snails and only one was proven to be effective. This fraction underwent
chemical analyses to define its chemical structure and functional groups. The results of
these analyses were as follows:
IR data
The IR spectrum confirmed a carbon–carbon double bond (ν1630 cm
−1
), C–H stretching
(ν2930 cm
−1
), ester carbonyl group (ν1553 cm
−1
), and revealed the presence of OH
(ν3400 cm
−1
broad) (figure 1(a)).
1
H-NMR data
1
H (300 MHz, CDCl
3
): 7.26 (2H, s) (H-2, H-6), 3.72 (3H, s) (COOCH
3
) as shown in
table 3and figure 1(b).
According to the previous data, the compound was identified as follows:
IUPAC name: 3,4,5-trihydroxybenzoate
Common name: Methyl gallate
Molecular formula:C
8
H
8
O
5
(figure 2).
The effect of sublethal concentrations on protein content of the snails’haemolymph
Filtrate of P. janthinellum
The protein bands separated from snails treated with LC
5
(0.1%), LC
15
(0.3%) and LC
25
(0.5%) were 14, 13 and 14 bands, respectively compared with 18 bands from the control
snails. The bands with molecular weights equal to 116.75, 45 and 40.455 KD disappeared
Separation of a compound effective against B. alexandrina 5
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in both groups of snails exposed to LC
15
and LC
25
. The 61.142 KD band disappeared in
both LC
5
and LC
15
treated snails. There were 7, 8 and 9 excess bands in snails treated
with LC
5
,LC
15
and LC
25
, respectively. The minimum similarity index was that of protein
bands separated from snails exposed to LC
25
(table 4and figure 3).
Methyl gallate
The number of protein bands separated from snails exposed to LC
5
(0.1%), LC
15
(0.3%)
and LC
25
(0.5%) were 12, 13 and 15 bands, respectively compared with 15 bands from
the control group. The 98.979 and 69.954 KD bands were shared between LC
15
and LC
25
treated snails. The 15.987 KD band was shared between LC
5
and LC
15
groups. The high-
est number of shared bands was recorded between control and LC
25
treated snails (similar-
ity index = 0.8). The minimum similarity index with control group was that of snails
treated with LC
5
as it equals 0.22 (table 5and figure 4).
The toxicity of sublethal concentrations to D. pulex
Exposure of D. pulex individuals to LC
25
of P. janthinellum filtrate resulted in only 10%
death. No mortality was observed in both LC
5
and control groups (table 6). Concerning
Table 1. The effective fungal species isolated from the surveyed governorates.
Fungal species
Number of
isolates Governorate Sample
Aspergillus niger var.
awamori
22 El-Giza, El-Ismailia, El-Gharbeya and El-
Menoufiya
Water and
soil
Aspergillus terreus 27 El-Giza, El-Ismailia, El-Gharbeya and El-
Menoufiya
Water and
soil
Aspergillus terreus var.
africanus
7 El-Giza Water
Aspergillus clavatus 17 El-Giza, El- Gharbeya and El- Menoufiya Water and
soil
Aspergillus flavus 15 El-Gharbeya and El- Menoufiya Water and
soil
Aspergillus petrakii 5 El-Gharbeya Soil
Aspergillus niveus 4 El-Gharbeya Soil
Aspergillus viridinutans 4 El-Ismailia Water
Aspergillus tamarii 11 El-Giza Water
Aspergillus oryzae 9 El-Giza Water
Aspergillus parasiticus 8 El-Giza Water
Acremonium alabamense 3 El-Ismailia Water
Acremonium spinosum 3 El-Gharbeya Soil
Fusarium proliferatum 7 El-Ismailia Soil
Trichoderma koningii 6 El-Gharbeya Soil
Rhizopus azygosporus 10 El-Giza Soil
Penicillium implicatum 14 El-Giza, El-Ismailia and El-Gharbeya Water
Penicillium janthinellum 10 El-Ismailia Water
Penicillium citrinum 12 El- Menoufiya Water
Microascus manginii 2 El-Gharbeya Soil
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the identified compound (methyl gallate), it was found that only 3.3% of the exposed
organisms died at LC
25
. No mortality was observed in both groups exposed to LC
5
and
LC
15
(table 6).
Table 2. LC
50
and LC
90
values of the effective fungal species on adult B. alexandrina snails after 24 h of
exposure.
Fungal species
Fungal concentration (%)
LC
50
LC
90
Aspergillus niger var. awamori 5.02 7
Aspergillus terreus 1.05 2.08
Aspergillus terreus var. africanus 2.07 4
Aspergillus clavatus 2.07 4
Aspergillus flavus 5.02 7
Aspergillus petrakii 4.06 8.08
Aspergillus niveus 5.08 7.02
Aspergillus viridinutans 5.02 7
Aspergillus tamarii 1.07 3.04
Aspergillus oryzae 5.02 7
Aspergillus parasiticus 7.04 8.02
Acremonium alabamense 4 5.08
Acremonium spinosum 5.02 7
Fusarium proliferatum 5.02 7
Trichoderma koningii 1.07 3.04
Rhizopus azygosporus 7.02 8.02
Penicillium implicatum 5.02 7
Penicillium janthinellum 1.03 4.09
Penicillium citrinum 6.08 8
Microascus manginii 2.08 4.05
Figure 1. Spectroscopic analyses of the effective compound from P. janthinellum; (a) IR spectroscopy and (b)
NMR spectroscopy.
Separation of a compound effective against B. alexandrina 7
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Discussion
The most frequent species in the present study were Aspergillus terreus,Aspergillus niger,
Aspergillus clavatus,Aspergillus flavus and Penicillium implicatum. This is in agreement
with the findings that the most prevalent species were A. flavus and A. niger from freshwa-
ter areas [32].Several authors stated that A. niger,A. flavus,Aspergillus fumigatus and
A. terreus were fairly common in Nile water [33,34]. The most prevalent species from
water and mud samples at Aswan Governorate were A. niger,Penicillium puberulum and
Trichoderma harzianum [35]. It was also postulated that A. niger,A. flavus,A. fumigatus
and A. terreus were the most frequent species in water samples collected from the River
Nile [36–38]. In another study of freshwater in India, A. niger,A. fumigatus,A. flavus and
A. versicolor were recorded as the commonest species, showing maximum percentage
frequency and contribution [39].
From the present survey 11 Aspergillus spp., 3 Penicillium spp., 2 Acremonium spp.,
Fusarium proliferatum, Trichoderma koningii, Rhizopus azygosporus and Microascus man-
ginii were found to be effective on B. alexandrina snails. It was reported that 46.7% of
Oncomelania hupensis snails died after 24 h exposure to 400 mg/l of alcoholic extract
from a certain strain of A. niger [7]. In addition, diethyl ether polar fraction (30 mg/l) from
exocellular broth of A. fumigatus showed the highest molluscicidal activity on O. hupensis
snails (100% mortality) [40]. Furthermore, amongst five genera of fungi, Acremonium,
Aspergillus, Penicillium, Fusarium and Trichoderma that were isolated from the land snail
Monacha cartusiana, the highest mortality rate (30%) of this snail was attained by
A. flavus [41].
Table 3.
1
H-NMR spectral data of the effective compound.
Position δ
H
(ppm)
1
2 7.26 (2H, s) (H-2, H-6)
3
4
5
6 7.26 (2H, s) (H-2, H-6)
7
8 3.72 (3H, s) (COOCH
3
)
OH
HO
COOCH3
OH
Figure 2. Chemical structure of methyl gallate.
8A.E.-H.A. Saad et al.
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Table 4. Effect of sublethal concentrations of P. janthinellum filtrate on protein pattern of B. alexandrina
haemolymph.
Bands
Marker Control
Penicillium janthinellum
LC
5
(0.1%) LC
15
(0.3%) LC
25
(0.5%)
Mol
W % Mol W % Mol W % Mol W % Mol W %
1 200 9.36 200 4.98 200 4.08 200 5.98
2
3
4 177.75 4.54
5 137.12 2.24
6
7
8 124.78 3.42
9
10 116.25 12.9 116.25 10.6 116.25 3.76
11
12
13 108.2 5.89 108.72 4.26 108.2 3.21 108.72 4.23
14 105.65 1.94 105.65 1.51 105.65 2.07
15
16 101.68 1.85
17 97.4 11.5 97.4 13.1 98.336 9.93 98.336 11.3
18
19 90.91 8.14
20 81.414 14.3 80.299 17.3
21 78.114 25.4
22
23 73.922 4.33
24 71.911 13.8
25 66.2 18.3 66.2 8.81 66.2 8.06 66.2 11.3
26
27 61.142 10.4 61.841 3.99 61.142 4.91
28
29 56.471 8.26
30 52.752 13.4
31 50.41 7.9
32 49.841 1.73 48.722 4.23
33 47.628 4.19
34 45 17.8 45 6.28 45 3.07 46.034 7.94
35
36 40.455 8.96 40.455 2.06 41.179 4.19
37 39.743 14.2
38 36.369 9.32
39
40 31 16.4 30.171 10.4 31.555 6.12
41 28.194 4.37
42 26.706 2.3
43 24.957 0.839
44 21.5 8.4 22.091 0.196
45 19.628 0.944 19.274 5.97
46 17.278 0.968 17.278 5.63
(Continued)
Separation of a compound effective against B. alexandrina 9
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The present study revealed that the minimum LC
50
value was that of P. janthinellum
(1.03%). Molluscicidal activity of P. janthinellum filtrate observed in the present work
could be attributed to secondary metabolites of such species. The genus Penicillium has
been recognised as a rich source of bioactive secondary metabolites [42]. Examples
include the anticancer berkelic acid from Penicillium sp., polyketides with HIV integrate
inhibitory activity from P.chrysogenum and the insecticidal paraherquamides H and I from
P. cluniae [43,44]. Chemical analysis was carried out on P. janthinellum filtrate in the cur-
rent study to determine the molluscicidally active metabolite. It was identified as methyl
gallate (phenolic compound). The toxicity of cashew nut shell liquid extract against the
golden snail (Pomacea canaliculata) was found to be caused by the presence of several
phenolic compounds [45]. It was reported that Anacardium occidentale, a plant rich in
phenolic compounds showed molluscicidal activity against B. glabrata [46]. Moreover, the
molluscicidal activity of leaves and rhizomes extracts of Iris pseudacorus plant against
Table 4. (Continued).
Bands
Marker Control
Penicillium janthinellum
LC
5
(0.1%) LC
15
(0.3%) LC
25
(0.5%)
Mol
W % Mol W % Mol W % Mol W % Mol W %
47 14.4 5.38 14.665 1.78 15.489 7.86 15.773 6.59 15.489 2.83
No. of
bands
18 14 13 14
Similarity
index
0.44 0.32 0.31
Figure 3. SDS–PAGE of haemolymph proteins of B. alexandrina snails treated with P. janthinellum filtrate.
M: marker, C: control,1P: LC
5
, 2P: LC
15
, 3P: LC
25
.
10 A.E.-H.A. Saad et al.
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Table 5. Effect of sublethal concentrations of methyl gallate on protein pattern of B. alexandrina haemolymph.
Bands
Marker Control
Methyl gallate
LC
5
LC
15
LC
25
Mol W % Mol W % Mol W % Mol W % Mol W %
1 205.79 4.02
2 200 9.36 200 3.41
3 150.32 1.92
4 116.25 12.9 115.32 1.67
5 113.94 4.77 114.86 3.69
6 108.57 3.73 107.27 2.45 108.13 3.25
7 106.41 1.41 105.13 1.49 105.98 1.57
8 103.87 1.75
9 99.778 12.4 98.979 9.54 98.979 12.4
10 97.4 11.5 97.792 7.56
11 86.031 12.5 87.226 11.5
12 84.853 21.7
13
14 80.299 23.5
15 78.114 10.2 78.114 9.59
16 69.954 8.12 69.954 8.96 69.954 9.82
17 66.2 18.3 66.2 11
18 63.421 7.03 63.421 7.97 64.104 5.16
19 61.413 7.5
20 57.586 6.33 58.835 8.83
21 55.169 5.66
22 53.422 6.09
23 51.179 7.46
24 50.633 8.48 49.031 8.76
25 47.478 4.13
26 46.471 8.52
27 45 17.8 45 7.59 45.975 4.85
28 41.768 15.6
(Continued)
Separation of a compound effective against B. alexandrina 11
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Table 5. (Continued).
Bands
Marker Control
Methyl gallate
LC
5
LC
15
LC
25
Mol W % Mol W % Mol W % Mol W % Mol W %
29 31 16.4 30.14 0.227
30 25.817 0.891 25.456 0.98 25.456 1.23
31 21.5 8.4
32 18.702 6.57 18.702 6.1 19.365 8.13 19.031 7.25
33 15.44 8.05 15.987 5.97 15.987 8.36 15.44 8.52
34 14.4 5.38
No of bands 15 12 13 15
Similarity index 0.22 0.5 0.8
12 A.E.-H.A. Saad et al.
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B. alexandrina snails was caused by the presence of gallic acid [47]. Meanwhile, the activ-
ity of 14 phenolic compounds against Bulinus truncatus was investigated; the most effec-
tive one was gallic acid compound with para-methoxy group (LC
50
= 3.60 ppm) [48]. In
addition, methyl gallate was shown to have a molluscicidal effect on B. truncatus snails
[49]. Recently, methyl gallate was isolated from the molluscicidal plant Callistemon vimi-
nalis [50].
The present investigation of the electrophoretic pattern of haemolymph proteins revealed
that there were differences in the ranges of molecular weights of bands among treated and
control snails. Moreover, some bands disappeared in the treated snails but were present in
control group and vice versa. These results were in accordance with the findings on the
effects of certain pesticides on the electrophoretic pattern of protein in Lymnaea stagnalis
snails [51].
In the present work, the minimum similarity index was that of protein bands separated
from the group exposed to LC
25
of P. janthinellum filtrate. It was reported that because of
the intoxication by the pesticides, most of the developmental stages of L. stagnalis snails
showed gradual decline; not only in the number of protein fractions but also in the
Figure 4. SDS–PAGE of haemolymph proteins of B. alexandrina snails treated with methyl gallate, M: marker,
C: control, 1M: LC
5
, 2M: LC
15
, 3M: LC
25
.
Table 6. Effect of sublethal concentrations of P. janthinellum and methyl gallate on mortality percentages of
D. pulex.
Daphnia pulex
Treatment
Penicillium janthinellum Methyl gallate
Control LC
5
LC
15
LC
25
LC
5
LC
15
LC
25
Number of animals tested 30 30 30 30 30 30 30
Number of dead animals 0 0 0 3 0 0 1
Percentage of mortality 0 0 0 10 0 0 3.3
Separation of a compound effective against B. alexandrina 13
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intensities of some of these fractions [51]. The alterations in the number of protein
fractions were caused by partial or total arrest in the transcription of mRNA which ulti-
mately affects the translation. That is why specific fractions were missed in the treated
snails [52]. Furthermore, it was demonstrated that the total protein of O. hupensis snails
treated with A. fumigatus was less than that of control [53].
The effect of sublethal concentrations of methyl gallate on protein content of snails was
carried out to determine the difference between the effect of the filtrate as a whole with all
its chemical contents and that of the effective fraction alone. It was revealed that methyl
gallate resulted in the reduction in number of bands than the filtrate of its producer. In
addition, the least similarity index was recorded especially at LC
5
. These findings indicate
that the effects of purified fractions are different from those of the entire filtrate which
may contain several chemical compounds (secondary metabolites). These compounds may
interfere with each other, resulting in either synergism or antagonism of their effects. It
was found that although the detrimental activity of tested culture filtrates is attributed
mainly to its major compounds, the synergistic or antagonistic effect of one compound
present in a minor percentage in a mixture which recorded such activity has to be
considered [54].
Cladocerans are ecologically very important members of freshwater invertebrates and
amongst them Daphnia spp.have been often used as test organisms for the ecotoxicologi-
cal monitoring of aquatic ecosystems [55]. In our study, sublethal concentrations of each
of P. janthinellum filtrate and methyl gallate resulted in zero or low mortality percentages.
Several studies were carried out to evaluate the toxicity of some chemical and plant mol-
luscicides against Daphnia. It was reported that the LC
50
value of Ambrosia maritima
extract on Daphnia magna was much higher than the molluscicidal concentration [56]. It
was also demonstrated that the toxicity of Agave attenuata to Daphnia sp. was lacking or
low [57]. Furthermore, certain molluscicidally active chemical derivatives were proven to
be nontoxic to D. magna [58].
Conclusion
The sub lethal concentrations LC
25
of P. janthinellum filtrate and LC
5
of methyl gallate,
showed detrimental effects on the protein pattern of treated snails. On the other hand, these
sub lethal concentrations showed mild effects on D. pulex. This indicates that they can be
safely applied without harming other water fauna. The chemical analysis of P. janthinellum
filtrate resulted in the purification and separation of a new compound from this species
which is methyl gallate. Thus, this compound can be synthesised later and commercially
manufactured to be applied for snail control.
Disclosure statement
No potential conflict of interest was reported by the authors.
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