Parasitol Res DOI 10.1007/s00436-014-4239-4

ORIGINAL PAPER

Development of microsatellite markers in Caryophyllaeus laticeps (Cestoda: Caryophyllidea), monozoic fish tapeworm, using next-generation sequencing approach Ivica Králová-Hromadová & Gabriel Minárik & Eva Bazsalovicsová & Peter Mikulíček & Alexandra Oravcová & Lenka Pálková & Vladimíra Hanzelová

Received: 25 September 2014 / Accepted: 14 November 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Caryophyllaeus laticeps (Pallas 1781) (Cestoda: Caryophyllidea) is a monozoic tapeworm of cyprinid fishes with a distribution area that includes Europe, most of the Palaearctic Asia and northern Africa. Broad geographic distribution, wide range of definitive fish hosts and recently revealed high morphological plasticity of the parasite, which is not in an agreement with molecular findings, make this species to be an interesting model for population biology studies. Microsatellites (short tandem repeat (STR) markers), as predominant markers for population genetics, were designed for C. laticeps using a next-generation sequencing (NGS) approach. Out of 165 marker candidates, 61 yielded PCR products of the expected size and in 25 of the candidates a declared repetitive motif was confirmed by Sanger sequencing. After the fragment analysis, six loci were proved to be polymorphic and tested for heterozygosity, Hardy-Weinberg

equilibrium and the presence of null alleles on 59 individuals coming from three geographically widely separated populations (Slovakia, Russia and UK). The number of alleles in particular loci and populations ranged from two to five. Significant deficit of heterozygotes and the presence of null alleles were found in one locus in all three populations. Other loci showed deviations from Hardy-Weinberg equilibrium and the presence of null alleles only in some populations. In spite of relatively low polymorphism and the potential presence of null alleles, newly developed microsatellites may be applied as suitable markers in population genetic studies of C. laticeps.

Ivica Králová-Hromadová and Gabriel Minárik equally contributed to this work.

Introduction

I. Králová-Hromadová (*) : E. Bazsalovicsová : V. Hanzelová Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 04001 Košice, Slovakia e-mail: [email protected] G. Minárik : A. Oravcová Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynská dolina B-2, 84215 Bratislava, Slovakia G. Minárik : L. Pálková Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Sasinkova 4, 81108 Bratislava, Slovakia G. Minárik Geneton Ltd., Ilkovičova 3, 84104 Bratislava, Slovakia P. Mikulíček Department of Zoology, Faculty of Natural Sciences, Comenius University, Mlynská dolina B-1, 84215 Bratislava, Slovakia

Keywords Population genetics . STR marker design . Non-segmented fish tapeworms . Caryophyllidea

Caryophyllidean tapeworms (Platyhelminthes: Eucestoda) represent a unique group of tapeworms having monopleuroid body plan with absence of internal and external proglotization and only a single set of reproductive organs. They belong to a widely distributed group of intestinal parasites of cypriniform and siluriform fishes occurring in all zoogeographical regions except for the Neotropics (Mackiewicz 1972, 1994). Caryophyllideans might play an important role in evolutionary history of Cestoda. Based on the phylogenetical analyses, they form the basal group of tapeworms being identified as either the sister group to true tapeworm (i.e. Eucestoda), or in a more derived position as a sister lineage to the segmented Diphyllobothriidea (Olson et al. 2001; Mackiewicz 2003; Brabec et al. 2006). The order Caryophyllidea van Beneden in Carus, 1863, is represented by four families; Balanotaeniidae Mackiewicz et

Parasitol Res

Blair, 1978; Lytocestidae Hunter, 1927; Caryophyllaeidae Leuckart, 1878; and Capingentidae Hunter, 1930. The most speciose family Caryophyllaeidae possesses 20 genera, including the genus Caryophyllaeus Gmelin, 1790, with C. laticeps, as the generic type species and one of the most common representatives of the genus. Even if more than 200 years passed since the discovery of this species, Caryophyllaeus laticeps still represents taxonomically complex taxon. C. laticeps has predominantly been found in freshwater bream, Abramis brama (L.) and white bream, Blicca bjoerkna (L.), but it rather frequently occurs in some other cyprinid fishes such as zope, Ballerus ballerus (L.); white-eyed bream, Ballerus sapa (Pallas); vimba bream, Vimba vimba (L.); Macedonian vimba, Vimba melanops (Heckel); common nase, Chondrostoma nasus (L.); and common carp, Cyprinus carpio (L.) (Akhmerov 1960; Žitňan 1968; Kennedy 1969; Anderson 1974, 1976; Chubb 1982; Dubinina 1987; Scholz 1989; Protasova et al. 1990; Macko et al. 1993; Moravec 2001; Jirsa et al. 2008). The geographical distribution of C. laticeps involves Europe, spanning in north–southern direction from Sweden (Milbrink 1975) towards Turkey (Aydoğdu et al. 2008), in west–eastern direction from the UK (Kennedy 1969; Anderson 1974) towards Russia (Protasova et al. 1990) and further in northern Africa, Egypt and Morocco (Khalil 1971). Bazsalovicsová et al. (2014) reported data on rather high morphological plasticity of C. laticeps and demonstrated the existence of several phenotypically diverse forms (morphotypes), mostly specific to their fish hosts. These authors first supposed that C. laticeps represents a complex of several not yet described “new” taxa. However, results by Bazsalovicsová et al. (2014) based on DNA sequence variation (mitochondrial cytochrome c oxidase subunit I and large subunit of the ribosomal DNA) did not support genetic divergence within these species and has rebutted initial hypothesis based on exclusively morphological principles. In C. laticeps, only one well-supported clade, further divided into several lineages associated with geography and particular fish host species, was identified (Bazsalovicsová et al. 2014). These lineages generally corresponded to five morphotypes, which are currently morphologically defined in detail by Hanzelová et al. (2014). Bazsalovicsová et al. (2014) pointed also out that a large range of morphological variability in tandem with a relatively low genetic variation makes the species delimitation based only on morphological criteria very difficult. A recently discovered conflict between the morphology and phylogenetic relatedness in C. laticeps complex designates this species to be an interesting model for population genetic studies. Microsatellites, or short tandem repeat (STR) markers, are suitable tools for resolving genetic interrelationships within and between populations of the particular species.

These highly polymorphic multilocus repeats are distributed throughout the genome and their unique molecular characteristics, such as a codominance, Mendelian inheritance and high allelic variation predestined them to be the popular markers in population genetics, paternity tests, forensic genetics, ecological and evolutionary studies (Goldstein and Schlötterer 2000). The aim of this study was to develop a set of the microsatellite markers in C. laticeps using next-generation sequencing approach in order to apply these markers in future population genetic studies of this parasite.

Materials and methods Material C laticeps specimens from three geographically widely separated European localities (Slovakia, Russia, UK), and fish hosts (white-eyed bream, freshwater bream, zope) were applied in different methodological steps (for details see Table 1). Tapeworms were isolated from fish intestine, washed several times in physiological solution immediately after dissection and fixed in 96 % ethanol. Preparation of template DNA for NGS analysis The DNA samples for NGS analysis were prepared from 20 C. laticeps individuals dissected from infected fish hosts (white-eyed bream) from Slovakia (Table 1). The genomic DNA was isolated using the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany), and the concentration and purity was measured spectrophotometrically by NanoDrop (NanoDrop Technologies, Inc., Wilmington, Delaware, USA). In order to increase the quality of DNA, it was finally subjected to DNA Clean & Concentrator TM -5 (Zymo Research, Freiburg, Germany) column purification. Finally, DNA obtained from 20 individuals was equimolarly mixed and pooled sample containing 2 μg of total DNA was send to GenoScreen (http://www.genoscreen.com; Lille, France) for commercial NGS service GenoSat®. NGS analysis by the GenoScreen The DNA was fragmented and enriched for repetitive sequences containing eight different repetitive motifs (TG, TC, AAC, AAG, AGG, ACG, ACAT, ACTC). The sequencing was performed on Roche GS/FLX Titanium sequencer. The QDD software (Meglécz et al. 2010) was used for identification of microsatellite marker candidates and design of corresponding primers.

Parasitol Res Table 1

Details on Caryophyllaeus laticeps individuals utilized in different methodological steps

Locality

Country

No. of samples Methodology

Purpose of the method

No. of microsatellite candidates

River Danube

Slovakia Ballerus sapa

20

Next-generation sequencing

Selection of suitable microsatellite loci candidates

165 after NGS and QDD analysis

Zemplínska šírava Slovakia Abramis brama River Danube Slovakia B. sapa

2 2

PCR Sequencing

Specific amplification and presence of declared STR motif

61 amplification positive 25 containing declared repetitive motif

River Danube Not specified River Volga River Danube Not specified River Volga

5 5 5 20 20 19

Fragment analysis Determination of polymorphic loci candidates

Slovakia UK Russia Slovakia UK Russia

Host

B. sapa A. brama Ballerus ballerus B. sapa A. brama B. ballerus

6 polymorphic loci

Fragment analysis Heterozygosity tests and calculation 5 suitable candidate loci Statistics of Hardy-Weinberg equilibrium

PCR amplification and sequencing The primers recommended by GenoScreen were applied on four C. laticeps individuals (Table 1) in order to test the PCR amplification effectiveness of designed markers. The total volume of PCR mixture was 20 μl and contained 10–20 ng of genomic DNA, 5 pmol of each of the two primers, 0.2 mM of each of the deoxynucleotide triphosphate (Fermentas, Vilnius, Lithania), 0.5 U of Taq DNA polymerase (Fermentas) with corresponding reaction buffer and 1.5 mM MgCl2. The amplification was performed in the Bio-Rad C1000™ thermal cycler programmed for 5 min at 94 °C as the initial step, followed by 30 cycles 1 min at 94 °C, 1 min at 55 °C and 2 min at 72 °C. The final step was 5 min at 72 °C. The PCR products were visualized on the 1 % agarose gel. Only loci with positive amplification of PCR products of expected size in all four individuals were considered for further sequencing procedure. In order to confirm the presence of declared repetitive motif, PCR products were sequenced from both sides using BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and ABI 3130xl Genetic Analyser (Applied Biosystems). The sequences obtained were visually checked for presence of declared repetitive motif using Geneious software version 6.0.5 (Biomatters, Auckland, New Zealand).

further analysis. On the other hand, loci that were proved to be polymorphic were tested by fragment analysis in the second step. For this purpose, the tested sampling set was increased to 59 individuals (the same localities as mentioned above; Table 1) and the results of fragment analysis were applied for tests of heterozygosity and Hardy-Weinberg equilibrium (HWE). For fragment analysis, 1 μl of amplified PCR product was mixed with 8.5 μl of HiDi Formamide (Applied Biosystems) and 0.5 μl of GeneScan-LIZ500 Size Standard (Applied Biosystems). Subsequently, the mix was denatured for 5 min at 94 °C and capillary electrophoresis on ABI 3130xl Genetic Analyser (Applied Biosystems) was performed. The GeneMapper v.3.7 software (Applied Biosystems) was used for genotyping. The selected polymorphic loci were tested for observed (Ho) and expected (He) heterozygosity, deviations from Hardy-Weinberg equilibrium and the presence of null alleles on 59 individuals coming from Slovakia (n=20), Russia (n=19) and UK (n=20) (Table 1). Heterozygosity and test for HWE were performed in GenAlEx 6.5 (Peakall and Smouse 2006, 2012). The presence of null alleles in selected loci and populations was estimated with Micro-Checker 2.2.3 (van Oosterhout et al. 2004).

Results Fragment analysis and population genetic statistics NGS analysis For determination of STR allele polymorphism of tested loci, fragment analysis with fluorescently labelled primers was performed in two steps. In the first step, samples of 15 C. laticeps individuals coming from Slovakia, Russia and the UK (Table 1) were tested for all candidate loci containing declared repetitive motif. Loci, which were detected to be monomorphic in all 15 tested individuals, were excluded from

The following results were obtained after NGS analysis from the GenoScreen service. The number of reads gained after filtering was 60,515. Of them, 7784 reads contained microsatellite motifs and 165 candidate microsatellite markers were identified. The identified microsatellite candidates belonged to di-, tri, tetra- and pentanucleotide repeats (30.3, 26.7, 41.8

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