Open Journal of Genetics, 2013, 3, 141-158 OJGen
http://dx.doi.org/10.4236/ojgen.2013.33017 Published Online September 2013 (http://www.scirp.org/journal/ojgen/)
Characterization of randomly amplified polymorphic DNA
(RAPD) fragments revealing clonal variability in cercariae
of avian schistosome Trichobilharzia szidati (Trematoda:
Schistosomatidae)
Anna Korsunenko1,2, Galina Chrisanfova1, Alexander Arifov1, Alexey Ryskov1,
Seraphima Semyenova1
1Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia
2Center of Parasitology, A.N. Severtsov Institute of Problems of Ecology and Evolution, Russian Academy of Sciences, Moscow,
Russia
Received 26 May 2013; revised 20 June 2013; accepted 8 July 2013
Copyright © 2013 Anna Korsunenko et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Recently we applied randomly amplified polymorphic
DNA (RAPD) fingerprinting to detect clonal variabil-
ity among individual cercariae within daughter spo-
rocysts and rediae of 10 digenean trematodes (Platy-
helminthes: Trematoda). The most variable RAPD
patterns were obtained for Schistosomatidae repre-
sentative—avian schistosome Trichobilharzia szidati.
In this work, 50 polymorphic DNA fragments of ap-
proximately 300 - 1500 bp from RAPD patterns of
individual T. szidati cercariae were cloned and se-
quenced. As a result genomic DNA sequences (total
length of approximately 41,000 bp) revealing clonal
variability in T. szidati cercariae were obtained and
analyzed. The analysis indicated that these sequences
contained tandem, inverted and dispersed repeats as
well as regions homological to retroelements of two
human parasites, Schistosoma mansoni and S. japoni-
cum. Tandem and inverted repeats constituted 8.9%
and 22.1% respectively, while the percentage of dis-
persed repeats was 21.0%. The average content of
these components was 41.7% with the average AT
content being 59.0%. About 40% of sequences in-
cluded regions ranging in length from 96 to 1005 bp
which displayed amino acid homology with open
reading frame pol products of S. mansoni and S. ja-
ponicum retroelements: non-long terminal repeat re-
trotransposons (nLTRs, 76%), long terminal repeat
retrotransposons (LTRs, 14%), and Penelope-like
elements (PLEs, 10%). Most of these regions (86.4%)
contained frameshifts, gaps, and stop-codons. The
largest portion of them was homological to nLTRs of
the RTE clade (67%). The number of sequences ho-
mologous to the members of CR1 lineage was 7 times
smaller (9%). Homology with LTRs of Gypsy/Ty3 and
BEL clades was revealed in 5% and 9% of cases re-
spectively. We assume that the repetitive elements
including retroelement-like sequences described in
the current study may serve as the source of clonal
variability detected previously in T. szidati and other
digenean trematodes. Such genome regions rapidly
accumulate mutations and thus may play an impor-
tant functional role in the life history of the species.
Keywords: RAPD Variability; Cercariae Heterogeneity;
Trichobilharzia szidati Repetitive DNA
1. INTRODUCTION
The vast majority of eukaryotic species reproduces bi-
sexually, yet approximately one out of every 1000 mul-
ticellular eukaryotic taxa is unisexual (parthenogenetic)
or asexual. Parthenogenetic reproduction occurs in many
phyla, especially in plants and invertebrates [1]. Among
invertebrates, the Digenea (Platyhelminthes: Trematoda)
have by far the most complex life cycles which usually
involve free-living and parasitic stages and always in-
corporates both parthenogenetic (within molluscan first
intermediate host) and sexual (within vertebrate defini-
tive host) reproduction. There are some stages in dige-
nean life cycle (parthenitae) reproducing via diploid par-
thenogenesis—mother sporocyst and either daughter
sporocysts or rediae [2,3]. The reproductive function in
parthenitae is performed by actively functioning ger-

A. Korsunenko et al. / Open Journal of Genetics 3 (2013) 141-158 142
minal masses [4]. Numerous free-swimming larvae (cer-
cariae) are formed in daughter sporocysts or rediae after
undergoing parthenogenetic reproduction. Since partheni-
tae are the result of diploid parthenogenesis with only
one parent involved, all cercariae forming within sporo-
cyst or redia might have been expected to represent a
group of genetically identical individuals—the clone. Fun-
ctionally diploid parthenogenesis can be viewed as a
simple cell division. Acquirement of such type of repro-
duction is an essential adaptation developed by trematodes
which allowed them to be evolutionary successful [2,3].
In general, genetic variation was described previously
for different invertebrate clonal systems using a variety
of molecular approaches [5]. Among digeneans, the vari-
able occurrence of W1 and W2 repetitive elements was
detected within and among daughter sporocyst genera-
tions of Schistosoma mansoni cultured in vitro [6,7].
Since an unexpected heterogeneity was found even among
clonal cercariae arising from monomiracidial snail infec-
tions the mitotic recombination events were suggested to
occur during the parthenogenetic reproduction of schis-
tosomes. The clonal variability was also detected in S.
mansoni [8] and S. japonicum [9] using multi-locus mi-
crosatellite analysis. In addition, RAPD variability with-
in daughter sporocysts was determined in Microphallus
pygmaeus and M. pseudopygmaeus [10].
RAPD-PCR is known as a DNA polymorphism analy-
sis based on the amplification of random DNA segments
with single primers of arbitrary nucleotide sequence,
usually 1 - 12 bp in length [11,12]. It detects DNA
polymorphisms produced by point mutations, insertions
or deletions in the genome. RAPD assay has a distinct
advantage of not requiring any specific nucleotide se-
quence information for amplification and can be em-
ployed across species using universal primers. This
method is targeted primarily to abundant sequences with-
in the genome, and usually generates a population of
amplification products that can be characteristic of a
specific organism. Because RAPD technique can reveal
considerable polymorphism even between closely related
organisms it has been used successfully for identification
and differentiation of various parasite species [13]. In
addition, RAPD-derived sequences obtained via cloning
of PCR products can be used to design more specific and
sensitive primers to develop locus-specific, or SCAR
(Sequence-Characterized Amplified Regions), markers
for parasite diagnosis. For example, using RAPD-derived
sequences we developed a specific primer pair to detect
three European causative agents of cercarial dermatitis in
humans (T. franki, T. szidati, and T. regenti) during the
prepatent period and after cercariae shedding [14].
Since RAPD assay is a method suitable for DNA
polymorphism detection in organisms containing even
small amounts of sample material we recently used this
technique to reveal clonal heterogeneity between indi-
vidual cercariae within sporocysts and rediae of 10 dige-
nean trematodes from Schistosomatidae, Strigeidae, Gor-
goderidae, Bucephalidae, Diplostomatidae, Plagiorchii-
dae, Halipegidae, Notocotylidae, and Echinostomatidae
families [15].
Here, we characterized RAPD fragments revealing
clonal variability between individual cercariae within
daughter sporocysts of the avian schistosome T. szidati
since the most variable RAPD patterns were obtained
previously for this Schistosomatidae representative [15].
The analysis of polymorphic DNA fragments included
sequence homology search using available nucleotide
and amino sequences of the avian (Trichobilharzia spp.)
and mammalian (S. mansoni and S. japonicum) schisto-
somes, search for tandem, inverted and dispersed repeats
as well as AT/GC ratio calculation. Such information
may contribute in determining structural genome organi-
zation of the avian schistosomes of Trichobilharzia spp.
which are the most frequent causative agents of cercarial
dermatitis in humans. RAPD-derived sequences of T.
szidati obtained in the current study can be also valuable
in cytogenetic studies. Our data may help to further in-
vestigate the mechanisms underlying clonal variability
detected in digenean trematodes.
2. MATERIALS AND METHODS
In total, 250 cercariae of T. szid ati were isolated from 47
daughter sporocysts sampled from nine naturally infected
snails of Lymnaea stagnalis. Four snails (Lsm1, Lsm2,
Lsm7, Lsm20) were collected from Russian (Moscow)
and five snails (NLst4, NLst9, NLst10, NLst11, Stag1)
from Belarusian (Naroch Lake) freshwater ponds. The
parasite species identification was resolved using inter-
mediate host-specificity combined with ITS2 rDNA, the
amplification reactions were performed by using the
primers its3Trem and ITS4 Trem [16].
Pieces of individual sporocysts were isolated from
snail hepatopancreas, and, after washing in distilled wa-
ter, fixed in 70% ethanol. The parthenitae were dissected
with a preparation needle to isolate three to six cercariae.
DNA was extracted from individual cercariae as de-
scribed previously [17].
Since DNA amount extracted from individual cercaria
is limited, the most intensely stained and unambiguous
RAPD profiles were obtained only when the DNA sam-
ple was used for no more than three primers. Among
tested random primers, those which detected the largest
number of polymorphic markers were selected for this
study. RAPD-PCR was performed with each of the cho-
sen 10-mer primers, P29 (5’-CCGGCCTTAC-3’),
OPA09 (5’-ACCGGACACT-3’), and OPA10
(5’-GTGATCGCAG-3’), in 25 µl volume containing 10
ng of total DNA, 75 mMTris-HCl (pH 8.8), 20 mM

A. Korsunenko et al. / Open Journal of Genetics 3 (2013) 141-158 143
centage of tandem, inverted and dispersed repeats as a
ratio of the number of nucleotide positions included in at
least one repeat to total length of analyzed sequence.
AT/GC ratio was calculated by DNA/RNA Base Compo-
sition Calculator software
(http://www.currentproto-cols.com/tools/dnarna-base-co
mposition-calcu lator).
(NH4)2SO4, 0.01% Tween 20, 5 mM MgCl2, 0.25 mM of
each dNTPs, 1 µM of primer, and 0.6 - 0.7 u of Taq DNA
polymerase (Fermentas, Vilnius, Lithuania). The follow-
ing amplification profile was used: 2 min of denaturation
at 95 C, 35 cycles of 1 min at 94 C, 1 min at 38 C, 15 sec
at 45 C and 2 min at 72 C, followed by the final 10 min
extension at 72 C. Reaction mixture containing no tem-
plate DNA was used as negative control for PCR assays. Using the Basic Local Alignment Search Tool (BLAST)
sequences of T. szidati were subjected to search against
the nucleotide (blastn) and protein (blastx) sequences
databases of schistosomes (S. mansoni, S. japonicum,
and Trichobilharzia spp.) available throughout the NCBI
(http://www.ncbi.nlm.nih.gov). Sequence similarities iden-
tified by the BLAST algorithms were considered statisti-
cally significant with E value of ≤10−5.
In total, 50 DNA fragments of approximately 300 -
1500 bp were cut and eluted from the agarose gel using
GFX PCR DNA and Gel Band Purification Kit (Amer-
sham, Piscataway, New Jersey) and cloned into the
pGEM-T Easy Vector (Promega, Madison, Wisconsin)
following the manufacturer’s instructions. Plasmid isola-
tion was made with GeneJET Plasmid Miniprep Kit
(Fermentas, Vilnius, Lithuania). For each RAPD frag-
ment one clone was selected for processing by the auto-
matic sequencing system ABI PRISM 3100-Avant (Ap-
plied Biosystems, Foster City, California). The nucleo-
tide sequences were deposited to GenBank: JX049928-
JX049977 (Supplementary Table 1).
3. RESULTS
To detect clonal variability among individual cercariae
within sporocysts of T. szidati we selected three random
primers (P29, OPA09, OPA10) which previously deter-
mined the largest number of polymorphic markers in
cercariae RAPD profiles [15]. These primers yielded a
total of 105 RAPD bands in the range of 220 - 2000 bp.
Repeats search within obtained T. szidati sequences
was performed using Tandem Repeat Finder [18], In-
verted Repeat Finder [19] and Spectral Repeat Finder
(http://www.imtech.res.in/raghava/srf/). As a result of
automatic repeats search, a specific nucleotide position
may be included by analysis software into more than one
repeat. So the calculation of an abundance of repeats
based on a sum of lengths of all repeats found by the
analysis software may produce an overrated result. To
overcome this, we created an algorithm which uses the
data from repeats search software and calculates the per-
A set of 50 bright, clear, intensively amplified polymor-
phic fragments of approximately 300 - 1500 bp was
chosen from cercariae banding patterns to construct T.
szidati DNA library (Supplementary Table 1). Among
them, 20 fragments were obtained using the P29 primer,
while 14 and 16 bands were obtained using the OPA09
and OPA10 primers respectively. Several cloned RAPD
fragments obtained with the P29 (Ts54-27, Ts88-83),
OPA09 (Ts52-50, Ts58-00), and OPA10 (Ts10-87,
Ts72-64) primers are shown as examples in Figure 1.
Figure 1. RAPD patterns of Trichobilharzia szidati cercariae obtained with the P29 (a), OPA09 (b), and OPA10 (c) primers.
Lane M—molecular size marker (100-bp ladder), sp—sporocyst; the cloned bands are indicated by the arrow.

A. Korsunenko et al. / Open Journal of Genetics 3 (2013) 141-158 144
For each RAPD fragment one clone containing DNA
insert was randomly selected for sequencing. In total, 50
sequences ranging in size from 281 to 1505 bp were ob-
tained. There was no similarity between the sequences.
The analysis indicated the prevalence of AT-bases
(59.0%). Repeats search revealed a number of tandem,
inverted, and dispersed repetitive components within
these sequences (Supplementary Table 2). For example,
one of the sequences (Ts54-27) contained a microsatellite
repeat (CA)10, while a 309-bp-long minisatellite repeat
was identified within another sequence (Ts59-00). This
repeat consisted of nine 35-bp monomers. Interestingly,
Ts49-66 included a 167-bp region similar to Sau3A
minisatellite repeat of T. ocellata and T. regenti. Supple-
mentary Table 3(a) gathers the information concerning
sizes of homology regions, their identity/similarity (I/S)
levels, and significance. In analyzed T. szidati sequences
tandem and inverted repeats constituted 8.9% and 22.1%
respectively, while the percentage of dispersed repeats
was 21.0%. Average repeats content turned out to be
41.7% of total sequence length.
Additionally, BLAST algorithms (blastn and blastx)
were used to find T. szidati sequence homology with
nucleotide and amino acid sequences of the avian
(Trichobilharzia spp.) and mammalian (S. mansoni, S.
japonicum) schistosomes available throughout the NCBI.
Based on nucleotide sequence similarity search (blastn)
all the sequences revealed regions ranging from 35 to
643 bp homological to numerous parts of S. mansoni and
S. japonicum genome. Similarity with available Tricho-
bilharzia spp. sequences was either not found or had no
significance (E > 10−5) except for the case of Ts49-66
described above. Translated nucleotide sequence similar-
ity search (blastx) detected 22 regions (fragments) within
19 T. szidati sequences which reveal homology with
open reading frame pol products (reverse transcriptase,
endonuclease, and integrase) of S. mansoni and S. ja-
ponicum retroelements from three major subclasses: long
terminal repeat retrotransposons (LTRs), non-long ter-
minal repeat retrotransposons (nLTRs), and Penelope-
like elements (PLEs) (Supplementary Table 3(b)). All
these regions had no similarity between each other at
amino acid level, consequently, they represented non-
overlapping regions homological to retroelements.
Among them, three homology regions were found in
Ts82-32, two regions were detected in Ts49-66, while the
other sequences revealed one region each (Supplemen-
tary Table 3(b)). Nineteen of 22 detected fragments
were found to be disrupted by gaps (sequences, con-
taining these regions: Ts49-66, Ts51-24, Ts51-49,
Ts53-51, Ts64-57, Ts72-64, Ts74-83, Ts76-85, Ts82-32),
stop-codons (sequences, containing these regions:
Ts48-65, Ts49-66, Ts51-49, Ts52-50, Ts64-57, Ts69-102,
Ts72-64, Ts74-83, Ts76-85, Ts82-32), and frameshifts
(sequences, containing these regions: Ts49-66, Ts51-24,
Ts53-51, Ts71-00) (Supplementary Table 3(b)).
Our data indicated the predominance of regions (n =
13) homological to the following nLTRs of the RTE
clade: SjCHGCS20 and SjR2 (one region in Ts51-82,
Ts71-00, Ts82-32, Ts85-90 and two in Ts49-66) as well
as SR3, Perere-3, and SjCHGCS19 (Ts52-50, Ts64-57,
Ts72-64, Ts74-83, Ts76-85, Ts82-32, Ts88-83) (Supple-
mentary Table 3(b)). Lengths of homology subregions
varied from 45 to 546 bp with I/S level ranging from
26/50% to 77/86%. Moreover, a 55-bp fragment homo-
logical to untranslated region of Perere-3 was found in
Ts47-78 (Supplementary Table 3(a)). In contrast, regions
homological to the members of CR1 clade were found to
be 2.5 times lower as compared to RTE. These regions
demonstrating homology with Perere, Perere-4, -7, and
Perere-2, -5, -6 were detected in Ts53-51 and Ts51-24
respectively (Supplementary Table 3(b)). Lengths of
homology subregions were 177 - 405 bp, while values of
I/S varied from 33/55% to 55/74%. Furthermore, a
132-bp-long region homological (I/S = 48/75%) to Per-
ere-8 was revealed in Ts48-65 (Supplementary Table
3(b)). Previously, the truncated copy of Perere-8 was
identified as CR1 member [20] whereas by examining a
full-length copy of this element the apurinic/apyrimidinic
endonuclease domain was detected which is characteris-
tic of the more modern lineages of nLTRs [21].
The most lengthy homology region (1005 bp) was de-
tected in Ts51-49 (Supplementary Table 3(b)). This re-
gion revealed homology (I/S = 54/68%) with SjCHGCS3-
retrotransposon from Gypsy/Ty3 clade of LTRs. Leng-
ths of subregions homological to the other members of
this lineage (Saci-5, SjCHGCS1, and SjCHGCS4) ranged
from 702 to 966 bp, while I/S varied from 27/42% to
57/71%. Two other T. szidati sequences (Ts48-00, Ts83-
28) contained regions homologous to BEL representa-
tives of LTRs (SjCHGCS16, Saci-1, etc.) (Supplemen-
tary Table 3(b)). Lengths of homology subregions were
96 - 252 bp with I/S varying in the range of 42/63% -
57/74%.
In total collection two regions demonstrated homology
with PLEs: 273-bp region homological (I/S = 33/55%) to
SjPenelope-2 was detected in Ts69-102, while Ts82-32
included 123-bp region similar to SjPenelope-2, -3, and
Perere-10 (Supplementary Table 3(b)).
The most complex structure of homology regions was
identified in Ts49-66 (Figure 2). This sequence con-
tained two regions homological to RTE members
(SjCHGCS20 and SjR2): located towards 5’-end subre-
gion I and II formed the first region, while the second
one (designated as VI) was located closer to 3’-end.
These two regions were homological to different parts
within amino acid sequences of SjCHGCS20 and SjR2:
the first region had homology with 856 - 913 and 1029 -

A. Korsunenko et al. / Open Journal of Genetics 3 (2013) 141-158 145
Figure 2. Location of homology regions in Ts49-66 nucleotide sequence of Trichobilharzia szidati. Subregion
I and II formed the first region homological to SjCHGCS20 and SjR2 retrotransposons, the second region is
designated as VI. Subregions III, IV, and V represented the region homological to Sau3A minisatellite repeat of
T. ocellata and T. regenti. Regions without any homology are indicated in black. Reading frames are indicated
as +1 and +2.
1086 aa respectively, while the second region had ho-
mology with 713 - 768 and 886 - 941 aa respectively.
Therefore, Ts49-66 included regions homological to dif-
ferent copies of SjCHGCS20 and SjR2. Finally, subre-
gions III, IV, and V (located in the central part of the
sequence) represented the region homological to Sau3A
minisatellite repeat of T. ocellata and T. regent i .
Besides that, computer-assisted sequence similarity
search revealed two regions homological at amino acid
level to S. mansoni regulatory elements. One of them
(288 bp) detected in Ts67-61 was homologous to zinc
finger transcription factor gli2 with I/S being 69/80%,
while the other one (177 bp) detected in Ts04-84 was
homological to transcription initiation factor tfiid with
I/S being 92/98% (Supplementary Table 3(c)).
In summary, a set of RAPD-derived sequences (total
length of approximately 41,000 bp) which reveal clonal
variability in T. szidati cercariae were obtained and ana-
lyzed. The analysis indicated that these sequences con-
tained tandem, inverted and dispersed repeats. The aver-
age content of these components was 41.7% with the
average AT content being 59.0%. About 40% of se-
quences included regions ranging in length from 96 to
1005 bp which displayed amino acid homology with pol
products of S. mansoni and S. japonicum retroelements:
nLTRs (76%), LTRs (14%), and PLEs (10%) (Figure 3).
Most of these regions (86.4%) contained frameshifts,
gaps, and stop-codons. The largest portion of them was
homological to nLTRs of the RTE clade (67%). The
number of sequences homologous to CR1 members was
7 times smaller (9%). Homology with LTRs of Gypsy/
Ty3 and BEL lineages was revealed in 5% and 9% of
cases respectively.
4. DISCUSSION
The aim of this study was the molecular characterization
of anonymous nuclear genome sequences produced by
RAPD genotyping and revealing clonal variability be-
tween individual cercariae within sporocysts of T. szi-
dati. The majority of RAPD-derived sequences belonged
to repetitive elements. Among them there were tandem,
inverted and dispersed repeats as well as regions homo-
logical to retroelements of two human parasites, S. man-
soni and S. japonicum. The complete genome sequences
of S. mansoni, S. japonicum, and S. haematobium were
published recently [22-24]. Over a third part of their ge-
nomes consists of the repetitive DNA including mobile
genetic elements (MGE), predominantly retrotransposons.
The average tandem, inverted and dispersed repeats con-
tent (41.7%) in T. szidati sequences was comparable with
total repetitive DNA constitution in S. mansoni (40.0%),
S. japonicum (40.1%), and S. haematobium (43.0%) ge-
nomes [22-24]. The AT-content prevalence (59.0%) was

A. Korsunenko et al. / Open Journal of Genetics 3 (2013) 141-158 146
Figure 3. Distribution of homology regions detected in Tricho-
bilharzia szidati nucleotide sequences by three retroelement
subclasses. RTE, CR1—clades of non-long terminal repeat
retrotransposons (nLTRs); Gypsy/Ty3, BEL—clades of long
terminal repeat retrotransposons (LTRs); PLEs—Penelope-like
elements.
detected in analyzed sequences of T. szidati as previously
was reported for S. mansoni, S. japonicum, and S.
haematobium (65.3%, 66.5%, and 65.7% respectively)
[22-24]. Almost one-half of T. szidati sequences in-
cluded fragments homological to retroelements of
mammalian schistosomes (nLTRs, LTRs, PLEs). The
largest portion of them (76%) demonstrated homology
with nLTRs. This type of MGE was shown to be pre-
dominant in S. mansoni and S. japonicum genomes [22,24].
Both blood flukes are known to contain numerous fami-
lies from the CR1 and RTE clades of nLTRs [21]. Like-
wise, most of homology regions detected in T. szidati
sequences were similar to RTE and CR1 members. The
elements from R2 lineage of nLTRs also occur in S.
mansoni and S. japonicum, but do not comprise a sig-
nificant fraction of their genomes [21]. Homologues of
R2 members were not found among analyzed T. szidati
DNA fragments. This may be explained by the low fre-
quency or general absence of R2 representatives in ge-
nome of this avian schistosome. Additionally, we de-
tected homologues of LTRs from two clades (Gypsy/Ty3
and BEL) common in mammalian schistosomes [22,24].
Among T. szidati sequences those homological to PLEs
were revealed as well. PLEs are not very abundant in
mammalian schistosomes, representing less than 2% of
bases in the shortgun reads of their genomes [21].
Retrotransposons and other MGE colonize the ge-
nomes of nearly every eukaryote and they play an im-
portant role in their genome structural organization and
evolution [25]. The genetic variability caused by MGE
ranges from changes in single nucleotides to changes in
the size and arrangement of whole genomes. Given that
the majority of new MGE insertions tend to be deleteri-
ous to host, different mechanisms have been developed
to mitigate the reduction in host fitness. MGE commonly
integrates into non-coding regions, for example, introns
to increase their probability of survival because of less
visibility to natural selection. However, MGE activity
can result in positive benefit to their hosts providing
positive selection on elements [26,27].
The genomes of most organisms carry tens to thou-
sands of retrotransposon copies. The majority of them is
non-functional and contains disabling mutations. The
coding sequences of such copies are highly degenerate,
cluttered with stop codons, frameshifts, and large indels
[28]. The mammalian schistosome genome also contains
defective copies of transposable elements. For example,
the coding sequences of different S. mansoni retrotrans-
posons found to be truncated or contain insertions of
related copies [20,29]. Furthermore, retrotransposons in
S. japonicum were represented by one to 793 intact cop-
ies and hundreds to thousands of partial copies [24]. Pre-
viously, the degenerated inactive copies were found to be
more abundant in heterochromatic regions than in the
euchromatin [30,31].
Potential negative effects of MGE on the fitness of
their hosts necessitate the development of strategies for
transposon control. One of the most important mecha-
nisms to regulate MGE expression rates and transposi-
tion frequencies is RNA interference (RNAi) pathway
which recognizes intracellular double-stranded RNA
(dsRNA). MGE may represent a source of dsRNA and
thus represent the RNAi target [32]. RNAi as an impor-
tant tool to elucidate gene function has been identified in
S. mansoni and S. japonicum [33,34]. RNA silencing
system is likely to be common in other flatworms, in-
cluding avian schistosomes.
Recently, analysis of S. japonicum endogenous short
interfering RNAs (siRNAs) detected that the majority of
them are transposable-elements-derived [35,36]. It was
discovered that such siRNAs in Drosophila were derived
from heterochromatic genomic loci which have previ-
ously been identified as master regulators of transposon
activity [37]. These loci contain numerous defective clus-
tered MGE copies (fragments of MGE as well as nested
copies). Likewise, described in this study T. szidati DNA
fragments homological to retroelements may act as a
source of siRNA to put down MGE expansion. The most
part of these regions (86.4%) contains frameshifts, stop-
codons and gaps. Nevertheless, these sequences possess
a genetic memory of MGE bursts in the evolutionary
history. Taking into account that transposable element
activity facilitates emergence of new genes, modifies
gene expression patterns and promotes chromosomal
rearrangements, MGE bursts can contribute to the evolu-
tion of lineage-specific traits that increase adaptability of
the species [38,39]. Recently, it was suggested that the
higher nLTRs content in S. mansoni (15.4%) in relation

A. Korsunenko et al. / Open Journal of Genetics 3 (2013) 141-158 147
to S. japonicum (8.3%) can be attributed to higher repre-
sentation of two retrotransposon families (SR2 and Per-
ere-3/SR3) of the RTE clade compared with the repre-
sentation of their closest relative families in S. japonicum
[21]. Considering the model of origin of African schis-
tosomes from a migrating ancestral species dwelling in
Asia it is proposed that bursts of SR2 and Perere-3/SR3
in S. mansoni would be a consequence of the selection of
parasite populations in a new environment during the
migration and speciation of blood flukes in Africa.
Likewise, the bursts of MGE transpositions may have
taken place in the evolutionary history of T. szidati stud-
ied in the present work.
As it was mentioned above, the germinal cells of
parthenitae (daughter sporocysts and rediae) undergo
mitotic division instead meiotic, which may have sug-
gested their genetic identity. The main cause of genetic
instability in this case is homologous recombination
during mitosis. Mitotic recombination can lead to either
reciprocal (mitotic crossing over) or non-reciprocal (gene
conversion) transfer of genetic material.
The frequency of mitotic crossing over in somatic
cells of the most of eukaryotes is quite low. For example,
spontaneous homologous recombination occurs at a rate
of 10−6 to 10−5 per cell cycle between repeated DNA se-
quences in mammalian cells [40]. In contrast, the fre-
quency of homologous recombination can be very high
in fertilized eggs (1/500) and embryonic cells (up to 10−1)
[41,42]. Likewise, the frequency of homologous recom-
bination in proliferating germinal cells of digenean tre-
matodes appears to be significantly high. The longevity
of the germinal cell proliferation period was found to vary
in different groups of trematodes [4]. Generative func-
tion is absent or weakly expressed in the mother sporo-
cysts of the most archaic and primitive trematodes (Fas-
ciolidae, Philophthalmidae, Cyclocoelidae, Notocotyli-
dae, Halipegidae, and many Echinostomatidae). In con-
trast, the long proliferation period of germinal cells dur-
ing the parthenitae development was found in more spe-
cialized groups (Sanguinicolidae, Schistosomatidae,
Bucephalidae, Diplostomatidae, Strigeidae, Plagiorchii-
dae) [3]. These findings are well supported by our data
obtained earlier [15] in that we found that species which
have more germinal cell proliferation cycles tend to
exhibit higher levels of clonal variability. Based on
RAPD markers we revealed significant genetic hetero-
geneity (the percentage of polymorphic loci (P) ranged
from 17.8% - 29.4%) between cercariae within daughter
sporocysts of five specialized forms of digenean trema-
todes (Schistosomatidae, Gorgoderidae, Bucephalidae,
Diplostomatidae, Plagiorchiidae). Additionally, the high
level of clonal variability between cercariae within dau-
ghter sporocysts of Bucephalidae representative was
confirmed using several snail-hosts in another study [43].
In contrast, the low level of clonal variability (P values
ranged in 5.2% - 6.5%) was determined between cer-
cariae within rediae of four archaic forms of digenean
trematodes (Halipegidae, Notocotylidae, and Echinosto-
matidae). The average percentage of polymorphic loci
for specialized forms was four times higher (25.4%)
compared with archaic forms (6.2%). So, the numerous
proliferation events of germinal cells in parthenitae of
the studied specialized digeneans may lead to increased
recombination frequency and consequent accumulation
of different genome rearrangements, while the low level
of clonal variability observed using RAPD markers in
studied archaic digeneans reflects the limited multiplica-
tion of germinal cells and reduced frequency of recom-
bination. The recombination frequency is also influenced
by the content and chromosome localization of recom-
bined sequences.
Taking into account the specificities of RAPD tech-
nique [11,12] and sequence analysis results obtained here
we supposed that rearrangements occur in moderate and
highly repetitive genome fraction of T. szidati. The
clonal heterogeneity detected previously in mammalian
schistosomes [6-9] was shown to be based on the repeti-
tive DNA variability. To compare sequence analysis re-
sults of T. szidati (the specialized digenean representa-
tive) for one of the archaic digenean representative
(Echinoparyphium aconiatum) two polymorphic RAPD
fragments which reveal clonal variability in cercariae
within rediae were cloned and sequenced by us previ-
ously (unpublished data). The obtained sequences of 441
bp and 667 bp contained regions homological at amino
acid level to pol products of S. mansoni retrotransposons.
One of these sequences (GenBank KC902786) revealed
homology with Saci-3, the member of Gypsy/Ty3 clade
of LTRs. The other one (GenBank KC902787) was
homological to Perere-5 from CR1 clade of nLTRs. So,
in comparison to T. szidati clonal variability in E. aco-
niatum cercariae also seemed to be associated with rear-
rangements in repetitive DNA including retroelement-
like sequences. Since an important consequence of mi-
totic recombination is homozygosis of heterozygous
markers, the increase of recombination frequency may be
considered as the evolutionary adaptation of more spe-
cialized and evolutionary advanced digeneans to prevent
the spatial distribution of MGE compared to the archaic
forms. However, comparative analysis of MGE abun-
dance in both specialized and archaic groups of digene-
ans is required to support this hypothesis.
5. ACKNOWLEDGEMENTS
This work received financial support from the Russian Foundation for
Basic Research (12-04-01153-a, 12-04-90034-Bel_a), President RF Pro-
gram of Leading Scientific Schools (S.S. -5233.2012.4), the Russian
Academy of Sciences Programs (Molecular and Cell Biology) of the

A. Korsunenko et al. / Open Journal of Genetics 3 (2013) 141-158 148
Presidium of the Russian Academy of Sciences and President Grants
for Government Support of Young Russian Scientists (MK-1026.
2012.4).
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SUPPLEMENTARY MATERIALS
Supplementary Table 1. The list of cloned variable RAPD fragments from Trichobilharzia szidati cercariae banding patterns ob-
tained with the P29, OPA09, and OPA10 primers.
No. Fragment (clone) identification GenBank Acc. Num.Length, bpRandom primer Snail-host
identification
Significant (E ≤ 10−5)
homology with protein
database sequences
1 Ts04-84 JX049957 683 OPA09 Lsm2 +
2 Ts09-86 JX049976 535 OPA10 Lsm20 –
3 Ts10-87 JX049972 572 OPA10 Lsm1 –
4 Ts11-88 JX049966 792 OPA10 NLst10 –
5 Ts12-89 JX049973 549 OPA10 Lsm2 –
6 Ts27-79 JX049943 887 P29 Lsm7 –
7 Ts28-80 JX049945 510 P29 Lsm20 –
8 Ts31-81 JX049959 589 OPA09 Lsm7 –
9 Ts47-20 JX049933 1039 P29 NLst10 –
10 Ts47-26 JX049951 435 OPA09 NLst10 +
11 Ts47-78 JX049963 1243 OPA10 NLst4 –
12 Ts48-00 JX049930 428 P29 NLst9 +
13 Ts48-65 JX049944 1420 P29 Lsm7 +
14 Ts49-00 JX049950 503 OPA09 NLst9 –
15 Ts49-66 JX049937 1312 P29 Stag1 +
16 Ts50-67 JX049939 1235 P29 Lsm1 –
17 Ts51-24 JX049961 1320 OPA09 Lsm20 +
18 Ts51-49 JX049977 1137 OPA10 Lsm20 +
19 Ts51-82 JX049975 1018 OPA10 Lsm7 +
20 Ts52-50 JX049956 1082 OPA09 Lsm1 +
21 Ts53-51 JX049967 1383 OPA10 NLst10 +
22 Ts54-27 JX049942 640 P29 NLst11 –
23 Ts54-69 JX049954 739 OPA09 Stag1 –
24 Ts55-28 JX049947 1320 P29 Lsm20 –
25 Ts56-00 JX049952 522 OPA09 NLst10 –
26 Ts57-00 JX049953 288 OPA09 NLst11 –
27 Ts58-00 JX049955 281 OPA09 Lsm1 –
28 Ts59-00 JX049932 299 P29 NLst10 –
29 Ts59-60 JX049969 1190 OPA10 NLst11 –
30 Ts64-57 JX049940 1210 P29 Lsm2 +

A. Korsunenko et al. / Open Journal of Genetics 3 (2013) 141-158 151
Continued
31 Ts65-58 JX049968 1172 OPA10 NLst11 –
32 Ts65-97 JX049958 758 OPA09 Lsm2 –
33 Ts67-61 JX049965 1124 OPA10 NLst9 +
34 Ts69-102 JX049949 492 OPA09 NLst9 +
35 Ts71-00 JX049928 372 P29 NLst4 +
36 Ts72-64 JX049971 1215 OPA10 Lsm1 +
37 Ts74-83 JX049946 957 P29 Lsm20 +
38 Ts76-85 JX049962 794 OPA10 NLst4 +
39 Ts80-49 JX049974 857 OPA10 Lsm7 –
40 Ts81-50 JX049964 689 OPA10 NLst9 –
41 Ts82-32 JX049941 1505 P29 Lsm2 +
42 Ts83-28 JX049934 412 P29 NLst11 +
43 Ts84-00 JX049938 385 P29 Lsm1 –
44 Ts85-90 JX049931 1252 P29 NLst9 +
45 Ts88-83 JX049935 718 P29 NLst11 +
46 Ts91-86 JX049929 755 P29 NLst4 –
47 Ts92-87 JX049936 719 P29 Stag1 –
48 Ts94-00 JX049960 723 OPA09 Lsm7 –
49 Ts96-00 JX049948 356 OPA09 NLst4 –
50 Ts101-00 JX049970 317 OPA10 Stag1 –

A. Korsunenko et al. / Open Journal of Genetics 3 (2013) 141-158 152
Supplementary Table 2. Tandem, inverted, and dispersed repeats content and AT/GC content in Trichobilharzia szidati sequences obtained using RAPD-PCR.

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Continued

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Supplementary Table 3. RAPD-derived sequences of Trichobilharzia szidati cercariae identified by matches to nucleotide (a) and protein (b, c) databases of Trichobilharzia spp., Schistosoma mansoni, and S. japonicum. nLTRs—non-long terminal repeat retrotransposons; LTRs—long terminal repeat retrotransposons; PLEs—Penelope-like elements. The unclear clade assignment of Perere-8 is designated by “?”.
(a)

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(b)

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Continued

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Continued

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(c)