Molluscan Research

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Characterisation of Pseudosuccinea columella

and Radix natalensis (Gastropoda: Lymnaeidae) in
Egypt using shell and molecular data

Yasser Dar, Said Amer, Rima Zein Eddine & Gilles Dreyfuss

To cite this article: Yasser Dar, Said Amer, Rima Zein Eddine & Gilles Dreyfuss (2016):
Characterisation of Pseudosuccinea columella and Radix natalensis (Gastropoda:
Lymnaeidae) in Egypt using shell and molecular data, Molluscan Research, DOI:
10.1080/13235818.2015.1064512

To link to this article: http://dx.doi.org/10.1080/13235818.2015.1064512

Published online: 22 Feb 2016.

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 MOLLUSCAN RESEARCH, 2016
http://dx.doi.org/10.1080/13235818.2015.1064512

Characterisation of Pseudosuccinea columella and Radix natalensis (Gastropoda:

Lymnaeidae) in Egypt using shell and molecular data
Yasser Dara,b, Said Amerb, Rima Zein Eddinec and Gilles Dreyfussc
a
Zoology Department, Faculty of Science, Tanta University, Egypt; bDepartment of Zoology, Faculty of Science, Kafr El-Sheikh University,
33516, Egypt; cLaboratory of Neuroparasitology INSERMU 1094, Faculty of Pharmacy, University of Limoges, France

ABSTRACT ARTICLE HISTORY

Pseudosuccinea columella and Radix natalensis live in the same habitat in Egypt and are Received 14 January 2015
important intermediate hosts of Fasciola hepatica and F. gigantica. Our study aimed to Final version received 23 May
characterise both snail species using molecular analysis and shell measurements. The ranges 2015
of morphometric parameters overlapped in the two lymnaeids, indicating that they do not
KEYWORDS
clearly differentiate the two species. PCR-sequence analysis of the nuclear ribosomal small Conchology; Fasciola host;
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subunit rRNA and the polymorphic mitochondrial cytochrome oxidase subunit 1 (CO1) genes snails; taxonomy
were used to determine the genetic identity and the potential diversity of the snails. Little
intrasequence variations were detected in the sequences of both gene loci, indicating the
potential homogeneity of lymnaeid populations in Egypt. Generated sequences of the
mitochondrial CO1 gene locus for R. natalensis showed obvious heterogeneity compared to
other sequences in GenBank. Molecular characterisation of these lymnaeids might help to
understand the snails’ biodiversity in a bid to control these populations and their related
diseases.

Introduction and G. truncatula could sustain the larval development

Snails of the family Lymnaeidae Rafinesque, 1815 are
air-breathing freshwater, hermaphrodite gastropods
with worldwide distribution. They serve as intermedi-
ate hosts of a wide range of medically and veterinarily
important digenean species, including liver flukes
(Fasciola spp.), blood flukes (Schistosoma spp.) and gas-
trointestinal flukes (Echinostoma spp. and Paramphisto-
mum spp.) (Bargues and Mas-Coma 1997; Degueurce
et al. 1999; Horak and Kolarova 2001; Mas-Coma et al.
2005); consequently, snail control measures are some-
times imposed. These freshwater snails could also be
considered as biomarkers of the occurrence and distri-
bution of fluke infections (Bargues et al. 2011a) in
endemic areas. Host–parasite relationships are
obviously versatile in different members of Lymnaei-
dae, with Lymnaea (Galba) truncatula (O.F. Müller,
1774) and Pseudosuccinea columella (Say, 1817) pre-
ferred intermediate hosts of Fasciola hepatica (Lin-
naeus, 1758), whereas Radix natalensis (Krauss, 1848)
and R. auricularia (Linnaeus, 1758) are hosts for Fasciola
gigantica (Cobbold, 1855) (Brown 1994; Dalton 1999).
Distribution of Fasciola Linnaeus, 1758 species can be
influenced by host snail distributions as, for instance,
F. gigantica in Africa and Asia by species of Radix
(Mas-Coma et al. 2005). This distribution pattern
might be disrupted by the introduction of either the
snail or the parasite into novel geographic localities.
For instance, R. natalensis from Egypt were infected
with a French isolate of F. hepatica (Dar et al. 2010),
of F. gigantica originating from China, Egypt and Mada-
gascar (Dar et al. 2003a, b).
Pseudosuccinea columella is of American origin and
this invasive species now has a global distribution
(Ponder 1975; Boray 1978; Boray et al. 1985; Cruz-
Reyes and Malek 1987; Brown 1994; McKown and
Ridley 1995; Mas-Coma et al. 2005; Gutierrez et al.
2011; Bargues et al. 2011b). Fasciola hepatica infections
were detected in P. columella in the field in Australia
(Boray et al. 1985), Egypt (Ahmed and Ramzy 1999),
Brazil (Coelho and Lima 2003), Argentina (Cucher
et al. 2006), and Cuba and the Caribbean area (Gutier-
rez et al. 2011), as well as in the laboratory under differ-
ent experimental conditions (Gutierrez et al. 2003;
Pointier et al. 2007; Vazquez et al. 2013; Dar et al.
2014). Comparably, R. natalensis has a wide distri-
bution, especially in Africa (Brown 1994).
Pseudosuccinea columella is characterised by spiral
ridges of the periostracum (Hubendick 1951; Brown
1994; Pointier et al. 2007) and a succiniform slender
shell with a long ovate shell aperture. Characteristically,
R. natalensis lacks the spiral threads on the periostra-
cum and has an ovoid shell with a wide body whorl
(Hubendick 1951; Brown 1994). However, classification
and nomenclature based on shell and anatomical fea-
tures are prone to considerable variability and do not
necessarily reflect evolutionary relationships (Bargues
et al. 2001; Correa et al. 2011). In recent years, molecular
analyses have been widely used in characterising

CONTACT Said Amer mssamer5@yahoo.com

© 2016 The Malacological Society of Australasia and the Society for the Study of Molluscan Diversity
 2 Y. DAR ET AL.

lymnaeid species and to infer their phylogenetic

0.57 ± 0.06
0.58 ± 0.04
0.66 ± 0.06
0.63 ± 0.05
0.61 ± 0.04
0.64 ± 0.04
0.62 ± 0.04
0.63 ± 0.04
aw/ah*
relationships. Moreover, genetic tools are used to
study the fluke/snail specificity and the role of geo-
graphic isolation and adaptation on such interaction

0.28 ± 0.05
0.27 ± 0.05
0.26 ± 0.04
0.27 ± 0.04

0.25 ± 0.04
0.24 ± 0.05
0.23 ± 0.05
0.23 ± 0.4
(Bargues and Mas-Coma 1997), as well as to infer the

sl/sh*
susceptibility or resistance to digenean infection

Ratios
(Gutierrez et al. 2003). Nuclear genes including small

0.72 ± 0.05
0.73 ± 0.05
0.74 ± 0.04
0.73 ± 0.04
0.77 ± 0.04
0.75 ± 0.04
0.76 ± 0.05
0.77 ± 0.05
subunit ribosomal RNA (18S rRNA), internal transcribed

ah/sh*
inter-spacers (ITS) (Bargues and Mas-Coma 1997;
Bargues et al. 1997; Remigio and Blair 1997a; Abouheif
et al. 1998; Stothard et al. 2000; Puslednik et al. 2009),

0.47 ± 0.04
0.50 ± 0.03
0.58 ± 0.07
0.54 ± 0.03
0.56 ± 0.04
0.55 ± 0.03
0.56 ± 0.03
0.57 ± 0.03
sw/sh*
and mitochondrial genes such as large subunit (16S)
(Remigio and Blair 1997b) and cytochrome oxidase
subunit 1 (CO1) (Bargues et al. 2011b) are widely

Spire length (sl)*

used in taxonomic and phylogenetic studies. Poly-

3.09 ± 0.72
2.68 ± 0.74
2.68 ± 0.65
2.74 ± 0.60
2.31 ± 0.54
2.73 ± 0.66
2.34 ± 0.60
2.45 ± 0.61
morphic loci such as microsatellite/minisatellite loci
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are frequently used to study the genetic diversity and

population structure of Lymnaeidae (Nicot et al. 2008).
In Egypt, both P. columella and R. natalensis occupy

Aperture width (aw)*

the same habitats and have considerable shell simi-
larity, especially in small specimens (Ahmed and

4.54 ± 0.81
4.21 ± 0.58
4.96 ± 0.78
4.64 ± 0.55
4.81 ± 0.60
5.23 ± 0.86
4.63 ± 0.44
5.24 ± 0.62
Table 1. Mean values (± SD) for shell measurements and ratios of Pseudosuccinea columella and Radix natalensis from Egypt.
Ramzy 1999; El-Shazly et al. 2002; Dar et al. 2005). In
addition, little is known about the genotypic identity
of these snails in Egypt. Therefore, this study was

Shell measurements (mm)

undertaken to determine the genetic identity of

Aperture height (ah)*

P. columella and R. natalensis along with the shell par-
ameters. To this end, lymnaeid species were deter-
8.03 ± 1.22
7.26 ± 0.82
7.51 ± 1.06
7.34 ± 0.83
7.90 ± 0.84
8.19 ± 1.32
7.45 ± 0.52
8.21 ± 0.68
mined by PCR-sequence analysis of the highly
conserved region of the nuclear ribosomal small
subunit rRNA (18S rRNA) gene and the potential diver-
sity was evaluated by the polymorphic mitochondrial
Shell width (sw)*

cytochrome oxidase subunit 1 (CO1) gene fragment.

Note: *Parameter showed significant difference (P < 0.001) between populations regardless of the species.
5.22 ± 0.93
4.96 ± 0.71
5.89 ± 0.97
5.49 ± 0.63
5.69 ± 0.67
6.04 ± 0.99
5.47 ± 0.49
6.09 ± 0.67

Materials and methods

Shell height (sh)*

Specimens of P. collumella and R. natalensis were col-

lected from small branches of the Nile River in different
11.11 ± 1.71

10.18 ± 1.47
10.08 ± 1.13
10.21 ± 1.07
10.91 ± 1.78

10.66 ± 0.98
9.93 ± 1.36

9.79 ± 0.92

places of the delta region in Egypt (Table 1), through

April 2012 to March 2013. A total of 40–50 specimens
were collected per site. Snails were transmitted to the
Population origin: city / governorate (GPS coordinates)

laboratory in open aerated aquaria. Snails were then

Kafr El-Zayat/Gharbia (30°49′ 31.68′′ N, 30°48′ 50.10′′ E)

fixed in 70% ethanol to perform shell morphometric

Khatatba/Monufia (30°22′ 24.73′′ N, 30°49′ 54.48′′ E)

Al-Wasta/Beni Suef (29°20′ 2.77′′ N, 31°12′ 17.83′′ E)

Toukh/Qalyubiyah (30°21′ 13.33′′ N, 31°12′ 7.39′′ E)

Toukh/Qalyubiyah (30°21′ 13.33′′ N, 31°12′ 7.39′′ E)

Al-Mahala/Gharbia (31°1′ 11.10′′ N, 31°5′ 26.37′′ E)
Al-Tahrir/Behaira (31°02 3.06 N, 30°27 39.00 E)

Mansouria/Giza (31°0′ 46.24′′ N, 31°22′ 51.89′′ E)

and molecular analyses.

′′

Soft parts of fixed snails were gently removed from

′

the shells which were washed several times with 70%

ethanol. Shells of each species were photographed
′′

using a stereomicroscope for a permanent record of

shell external features. Shells were then dehydrated
′

in a graded series of ethanol. Each shell was dried in

a critical point dryer, mounted on metal stubs, coated
with gold and examined with a JEOL1 HSU-5300 scan-
ning electron microscope (SEM) (JEOL Ltd.) at an accel-
erating voltage of 30 KV (Gabriel 1982).
Five measurements were determined for each snail
Snail species

R. natalensis
P. columella

shell: shell height (sh); shell width (sw); aperture

height (ah); aperture width (aw); and spire length (sl).
Measurements were done using a caliper and
 MOLLUSCAN RESEARCH 3

expressed in mm. Four ratios were also calculated for
each shell: sw/sh; ah/sh; sl/sh; and aw/ah (Table 1).
DNA was extracted from representative samples
(five samples for P. collumella and 12 for R. natalensis)
of each snail species using QiAamp DNA Mini Kit
(Qiagen) following the manufacturer’s instructions. A
Nanodrop ND-1000 spectrophotometer was used to
quantify and check purity of DNA. PCR amplification
of ribosomal small subunit (18S) rRNA and mitochon-
drial cytochrome c oxidase subunit I (CO1) were done
utilising primer sets described by Jorgensen et al.
(2010) and Correa et al. (2011), respectively. PCR reac-
tions were performed in 25 µl reaction volumes con-
taining 500 ng/µL of genomic DNA. The PCR mixture
contained 5 µl 10× buffer, 0.8 µl dNTPs (each), 0.5 µl
of each primer (20 ng/µL), 0.4 µl of Taq polymerase,
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to find variables characterising snail species and popu-
lations. Stepwise linear discriminant analysis was per-
formed to select the different variables. Calculations
were made using the software R (Ida and fda functions).
Results
Morphometric study
The two species have a similar shell shape (Figure 1),
but SEM examination of P. collumella shell surface
showed characteristic transverse microsculptures on
the periostracum that clearly distinguish this snail
from R. natalensis (Figure 2) which lacks these striations.
The morphometric study showed that the mean
values of R. natalensis shell width and aperture width

1.5 µl MgCl2. PCR conditions for 18S were set as a dena- were significantly larger (t = −6.951, P < 0.001; t =
turation step at 94 °C for 3 min, followed by 30 cycles of −6.234, P < 0.001, respectively) than those of
denaturation at 94 °C for 30 s, annealing at 55 °C for 30 P. columella. In addition, ratios sw/sh (W = 906.5, P <
s and extension at 72 °C for 45 s; a final extension step 0.001), ah/sh (t = −4.815, P < 0.001) and aw/ah (t =
at 72 °C for 7 min was included. Cycling conditions for
CO1 involved a denaturation step at 95 °C for 5 min, 30
cycles each including denaturation at 95 °C for 1 min,
annealing at 50 °C for 30 s and extension at 72 °C for
30 s, and a final extension step at 72 °C for 10 min. Pro-
ducts were subjected to electrophoretic separation
using 1.5% agarose gels, stained with ethidium
bromide and visualised on a UV transilluminator.
PCR products were purified using QIAquick PCR Puri-
fication Kit (Qiagen), according to the manufacturer’s
instructions and sequenced directly using Big Dye Ter-
minator v3.1 Cycle Sequencing Kit (Applied Biosys-
tems) on an ABI 3130 Genetic Analyzer (Applied
Biosystems). Generated sequences were assembled
using ChromasPro v1.5 software (http://technelysium.
com.au/?page_id=27). The obtained sequences were
aligned with each other and reference sequences
using ClustalX (http://www.clustal.org/) to confirm
species identification. Maximum likelihood (ML) with
GTR + G + I model implemented in MEGA5 (http://
www.megasoftware.net) was used to assess the phylo-
genetic relationships among different populations of
lymnaeids. Unique nucleotide sequences generated
in this study were deposited in GenBank under acces-
sion numbers LC015496–LC015509 for 18S and
LC015510–LC015523 for CO1.
Values of morphometric parameters and corre-
sponding ratios were subjected to comparison of var-
iances using Fisher’s exact test. When the variances
were equal, Student’s t-test was used; if not a Wilcoxon
test was applied to determine the levels of significance
between the two snail species. For analysis of snail
populations, the Kruskal and Wallis test was used if
Figure 1. Views of the shells of Pseudosuccinea columella from
the variances were different and one-way ANOVA
Al-Tahrir, Behaira governorate, Egypt and Radix natalensis from
when the variances were equal. All analyses were per- Mansouria, Giza governorate, Egypt. A, Dorsal view of
formed using Statview 5.0 software (SAS Institute Inc.). P. columella; B, aperture view of P. columella; C, dorsal view
In addition, the function discriminant analysis was used of R. natalensis; D, aperture view of R. natalensis.
 4 Y. DAR ET AL.
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Figure 2. SEM of shell surface. A, Pseudosuccinea columella; B, Radix natalensis. Scale bar = 500 µm.
−9.6, P < 0.001) were higher in R. natalensis than in
P. columella. In contrast, the spire length and the ratio
sl/sh of the former snail had significantly lower values
(W = 11408, P < 0.001, t = 4.815, P < 0.001, respectively)
than the latter. When the snail populations were com-
pared to each other, regardless of the species, signifi-
cant differences were noted between the populations
for all studied measurements and ratios (Table 1). Fur-
thermore, results of the function discriminant analysis
showed that three morphometric parameters (shell
height, aperture width and sw/sh ratio) could correctly
discriminate between the two snail species 90% of the
time. Additionally, the morphometric parameters used
in the present study could discriminate the snail popu-
lations of each species, but at a lower percentage
(50%). However, more samples were needed to ascer-
tain the utility of such parameters to discriminate
between snail populations.
Molecular analysis
Homology search of 18S sequences confirmed the
morphological identifications of the Egyptian snails as
P. columella and R. natalensis. Alignment of the
obtained sequences showed no intrasequence vari-
ations of both species as well as complete identity
with corresponding sequences in database.
CO1 sequences generated from P. columella snails
were identical to sequence FN598165 of P. columella
from Colombia (Bargues et al. 2011b) and AY227366
reported by Remigio and Hebert (2003) for specimens
from Australia and 98% identical to JN872458 of
P. columella from Argentina (Standley et al. 2013).
Notably, two sequence types of CO1 were generated
from R. natalensis, resulting in the occurrence of two
haplotypes (Rnh1 consists of seven individuals and
Rnh2 consists of five individuals). However, haplotype
codes were only provisional due to incomplete
sequences of the gene . The nucleotide polymorphism
of A in first sequence to G in the second was detected
at position 41. Moreover, marked heterogeneity was
noted when aligned with reference sequences in
GenBank, with 97% homology compared to
EU818835 of R. natalensis from Malawi (Albrecht et al.
2008) and 88% homology with sequence JN614403
for R. natalesis from Réunion (Correa et al. 2011).
Phylogenetic analysis based on CO1 sequences
showed that R. natalensis was toward the end of the
clades, and samples were separated into their respect-
ive sites. The Egyptian haplotypes of R. natalensis
clustered together with high bootstrap values of
100% (Figure 3) and with that from Malawi
(EU818835). In addition, our tree placed P. columella
and ‘Lymnaea’ diaphana King, 1830 as sister groups.
Discussion
Lymnaeidae is an important family because of its veter-
inary and public health significance in its role in digen-
ean transmission (Mas-Coma et al. 2005). Egypt is the
land bridge between Africa and Asia, so lymnaeids in
Egypt may have played a central role in dissemination
of trematode infections to other continents (Mas-Coma
et al. 2009). In this country, P. columella and R. natalensis
are important fascioliasis transmitting lymnaeids (Dar
et al. 2005). Analysis of shell measurements indicated
that the mean values of shell and aperture width, and
the ratios sw/sh, ah/sh and aw/ah, were larger in
R. natalensis compared to P. columella. Conversely,
R. natalensis had lower means for spire length and sl/
sh ratio than P. columella. These results agree with
the morphological features of both species (Figure 1)
as R. natalensis has a wide ovoid shell and
P. columella has a slender shell with a long ovate aper-
ture (Hubendick 1951; Brown 1994). However, the
range values of individual parameters overlapped in
 MOLLUSCAN RESEARCH 5
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Figure 3. Maximum likelihood (ML) tree showing the genetic relationship of Egyptian Lymnaeidae to others in GenBank based on
mitochondrial CO1 sequences. Evolutionary relationships of 30 taxa were inferred using ML (see Materials and methods). Numbers
at the internodes correspond to percent bootstrap values from 3000 replicates. Biomphalaria pfeifferi Krauss, 1848 (DQ084830) (Pla-
norbidae) and Trimusculus afra Gmelin, 1790 (EF489388) (Trimusculidae) were used as outgroups. Scale bar indicates 5 substitutions
per 100 sites.

the two species, indicating that these criteria alone
were not reliable in discriminating between them. For
instance, the shell width of R. natalensis varied from
8.7 mm to 4 mm, compared to 7.6 mm to 3.5 mm in
P. columella. Similarly, Bargues et al. (2011b) reported
that although the morphometric comparison allows
the differentiation of Galba cousini Jousseaume, 1887
and Lymnaea meridensis Bargues, Artigas & Mas-
Coma, 2011, the shell parameters of Galba cousini
and P. columella overlapped. In addition, Correa et al.
(2011) indicated that shell characters alone did not
reliably enable accurate identification of most Neotro-
pic lymnaeids. However, the previous authors restrict
the usage of shell shape to identify some species
which have distinguishing morphological characters
such as ‘L.’ diaphana, Galba cousini and P. columella.
This difficulty might be due to the phenotypic plasticity
in shell shape in lymnaeids, as their shells may vary
according to environmental conditions (Samadi et al.
2000; Pfenninger et al. 2006). Taken together, the
high intraspecific variability in shell morphology and
their anatomy may be insufficient to develop a robust
classification base for Lymnaeidae (Correa et al. 2011).
Thus, in addition to traditional morphological traits,
the characteristic micro-sculptures on the periostracum
(Figure 2) seem to be the most efficient character to
distinguish P. columella from other lymnaeids (Huben-
dick 1951; Brown 1994; Pointier et al. 2007; Correa
et al. 2011). However, this distinctive character is of
limited practical use for a non-expert malacologist
(Correa et al. 2011).
DNA sequences of 18S indicated no intrasequence
variation and the generated sequences were identical
to the corresponding sequences in GenBank, indicating
the conservative nature of the 18S gene. The conserva-
tive nature of SSU sequences has shown its suitability for
lymnaeid identification (Bargues and Mas-Coma 1997;
Bargues et al. 1997). In contrast, Stothard et al. (2000)
detected variation within and between populations of.
R. natalensis from Madagascar based on analysis of
SSU gene locus, reflecting the impact of selecting differ-
ent regions of the gene. Similarly, no intrasequence
polymorphism was detected in the generated mtDNA
CO1 sequences of P. columella and very little in those
of R. natalensis, indicating the potential genetic hom-
ogeneity (Bargues et al. 2011b) of Egyptian populations.
In contrast, two of the endemic clades of Radix spp. on
the Tibetan Plateau, China, showed a remarkably high
genetic diversity, perhaps due to multiple colonisation
events combined with a relatively long intra-plateau
 6 Y. DAR ET AL.
evolution (von Oheimb et al. 2011). However, obvious
genetic heterogeneity of R. natalensis CO1 sequences
in Egypt compared to reference sequences in databases
may indicate unique and uniform evolutionary events
for Radix populations in Egypt. Similarly, Bargues et al.
(2011b) reported that the considerable CO1 differences
between the lymnaeids from Réunion, Colombia and
Puerto Rico may indicate that two well separated
groups are involved. The present work is a pilot study
which cannot reach a final conclusion on the genetic
identity of lymnaeid populations in Egypt in the light
of the limited number of analysed samples, number of
genetic loci and the limitation of geographic locations
of sampling. Work is underway on a larger study
which addresses these limitations and covers a larger
number of lymnaeid species.
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Acknowledgements
The authors are very grateful to Dr Daniel Rondelaud, Faculty
of Pharmacy, University of Limoges, France, for critical revi-
sion of the manuscript and Dr Philippe Vignoles, Faculty of
Pharmacy, University of Limoges, France, for his assistance
in the statistical analyses. Thanks are also due to the anon-
ymous reviewers for their constructive and informative com-
ments which improved the quality of this paper.
Disclosure statement
No potential conflict of interest was reported by the authors.
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