Parasitology International 64 (2015) 243–250

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Pomphorhynchus laevis (Acanthocephala) from the Sava River basin: New insights into strain formation, mtDNA-like sequences and dynamics of infection Irena Vardić Smrzlić a,⁎, Damir Valić a, Damir Kapetanović a, Vlatka Filipović Marijić b, Emil Gjurčević c, Emin Teskeredžić a a b c

Laboratory for Aquaculture and Pathology of Aquatic Organisms, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb, Croatia Laboratory for Biological Effects of Metals, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb, Croatia Department for Biology and Pathology of Fish and Bees, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia

a r t i c l e

i n f o

Article history: Received 28 November 2014 Received in revised form 17 February 2015 Accepted 18 February 2015 Available online 26 February 2015 Keywords: Acanthocephalans Pomphorhynchus laevis Squalius cephalus Sutla/Sava Rivers Genetic variability Mitochondrial-like sequences

a b s t r a c t Here we report the genetic variability and presence of mtDNA-like sequences of Pomphorhynchus laevis from the chub, Squalius cephalus, caught at the sampling sites along the Sava River and its tributary the Sutla River in Croatia. Sequences of the cytochrome c oxidase subunit 1 (COI) gene of the recovered P. laevis specimens were used for haplotype network construction and phylogenetic analysis. These analyses showed that some specimens contained mitochondrial-like sequences, and they uncovered the existence of a Sava River basin strain different from known strains of P. laevis. This is the first time that P. laevis has been shown to contain mtDNA-like sequences, suggesting the need to exercise caution during COI analyses of P. laevis using universal primers. Highly conserved sequences of two nuclear markers, the ITS region and 18S rRNA, were not helpful for understanding genetic variability or differentiating strains. Furthermore, analysis of the dynamics of P. laevis infections in S. cephalus from the Sava and Sutla Rivers showed decreased prevalence and abundance at sites with inferior water quality, positive association of parasite abundance with fish size, and no clear association of parasite abundance with fish condition index or sex. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Pomphorhynchus laevis is the most abundant and widely distributed acanthocephalan species of freshwater fish in Europe [1]. Its preferred definitive hosts in fresh water are Barbus barbus and Squalius cephalus, and its intermediate host is the local species of freshwater amphipod of the genus Gammarus. This is believed to be the original, post-glacial pattern of host usage [2]. High intraspecific variability, both morphological [1,3–10] and genetic [1,7,10–13] has led to longstanding debates about whether different forms correspond to different species, as well as about the different strains of P. laevis. The current thinking based on morphological and molecular analyses of populations from continental Europe is that there are two cryptic species within the P. laevis complex: P. laevis sensu stricto and Pomphorhynchus tereticollis [1,10]. Investigations of P. laevis in the British Isles based on both morphological and molecular analyses confirmed the presence of three strains within the

⁎ Corresponding author. Tel.: +385 1 4561 076; fax: +385 1 4680 943. E-mail address: [email protected] (I. Vardić Smrzlić).

http://dx.doi.org/10.1016/j.parint.2015.02.004 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved.

P. laevis sensu stricto species, two freshwater ones and a marine one [9,12,14]. The Sava River is the largest tributary to the Danube River Basin [15] and was not included in previous studies of P. laevis genetic variability. Nevertheless, it has been hypothesised to be the source of the ancestral strain of continental P. laevis [12]. The aim of the present study was to examine the genetic variability of P. laevis from its original, post-glacial definitive host European chub (S. cephalus), from the Croatian part of the Sava River and its left tributary, the Sutla River. COI sequences were analysed to check for possible existence of mitochondrial DNA (mtDNA)-like sequences and for the existence of a P. laevis strain from the Sava River basin that is different from strains already described from continental Europe. In addition, the sequences of the two nuclear-encoded regions, the 18S rRNA gene and the ribosomal internal transcribed spacers ITS1 and ITS2, were analysed to gain molecular insights into the genetic variability of P. laevis from the Sava River basin. The dynamics of P. laevis infections were described with respect to the seasonal and spatial distribution of infection, as well as its association with host length, condition and sex.

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2. Materials and methods

Table 2 Prevalence and abundance of Pomphorhynchus laevis infection in chub, Squalius cephalus, sampled in the Sutla River, Croatia.

2.1. Sampling

Sutla River

Parasitological examination of P. laevis (Pomphorhynchidae, Palaeacanthocephala) specimens was performed on European chub (S. cephalus, Cyprinidae, Actinopterygii) sampled in the Sava River (266 fish) in four periods during 2005 and 2006 (Table 1), and in the Sutla River (172 fish) in two periods during 2008 and 2009 (Table 2) according to Croatian standard methods (HRN EN 14011 2005). Geographical positions of sampling sites are shown in Tables 1 and 2. The prevalence and abundance of P. laevis infections in the chub were calculated [16]. Standard length, weight and sex of chub were determined and Fulton's condition index (FCI) was calculated [17]. Fish were aseptically dissected and P. laevis specimens were collected, fixed in 75% ethanol or formalin for morphological determination, and stored at −80 °C for molecular analyses. Fixed P. laevis specimens were cleared in methyl salicylate and identified to genus level by light microscopy [18].

2.2. Molecular analyses 2.2.1. A COI marker We wished to check for the existence of mtDNA-like sequences because the occurrence of COI pseudogenes was reported in the genus Acanthocephalus as a result of the use of universal COI primers [19]. COI products were amplified by polymerase chain reaction (PCR), followed by sequencing and single-strand conformation polymorphism (SSCP) analysis. Total DNA was extracted from a single acanthocephalan using a DNA purification kit (DNeasy Blood and Tissue Kit, Qiagen) according to the manufacturer's instructions. PCR reactions (50 μl) were performed with the following mixture: 1× PCR buffer, 2.5 mM MgCl2, 0.4 mM dNTPs, 20 pmol of each primer, 1 U of Taq polymerase (Applied Biosystems), 50 ng of total DNA and water (Molecular Biology Reagent, Sigma). Universal primers used to amplify COI regions were described previously [20]. Reaction conditions were done as follows: 10 min at 94 °C (initial denaturation), 35 cycles of 30 s at 94 °C (denaturation), 45 s at 56 °C (annealing) and 1 min at 72 °C (extension), with a final elongation step of 10 min at 72 °C. Sequencing (Macrogen, Netherlands)

Table 1 Prevalence and abundance of Pomphorhynchus laevis infection in chub, Squalius cephalus, sampled in the Sava River, Croatia. Sava River Sampling site

S1. Otok Samoborski N 45°50,543′ E 15°43,497′ S2. Jarun N 45°46,572′ E 15°56,524′ S3. Oborovo N 45°41,286′ E 16°14,875′ S4. Lukavec Posavski N 45°24,081′ E 16°32,337′ S5. Jasenovac N 45°15,825′ E 16°53,658′ All sites

Parasite prevalence (%) Median abundance (min–max number of parasites) [Number of sampled fish] Spring 2005

Autumn 2005

Spring 2006

Autumn 2006

86.7 – [15] 56.3 – [16] 100 – [9] 30 – [10]

77.2 – [22] 60 – [15] 80 – [15] 46.7 – [15] 70 – [10] 67.5 – [77]

73.3 2.8 (0–12) [15] 59.1 4.3 (0–18) [22] 61.5 2.1 (0–5) [13] 46.7 1 (0–4) [15] 20.0 0.4 (0–3) [15] 52.5 2.3 (0–18) [80]

60.0 3.5 (0–15) [15] 71.4 1.7 (0–5) [14] 40.0 0.7 (0–2) [10] 30.0 0.5 (0–2) [10] 40.0 0.5 (0–2) [10] 50.8 1.6 (0–15) [59]

– 68.0 – [50]

Sampling site

S1. Hum N 46°13,339′ E 15°43,187 S2. Donje Brezno N 46°11,532′ E 15°39,269′ S3. Kumrovec N 46°07,830′ E 15°37,020′ S4. Klanjec N 46°02,728′ E 15°44,237′ S5. Kraj Donji N 45°54,602′ E 15°41,517′ S6. Ključ Brdovečki N 45°52,433′ E 15°41,426′ All sites

Parasite prevalence (%) Median abundance (min–max number of parasites) [Number of sampled fish] Autumn 2008

Autumn 2009

76.0 2.5 (0–68) [25] 0 0 (0–0) [6] 33.3 0 (0–3) [3] 83.9 2.0 (0–59) [31] 45.5 0 (0–4) [11] 100 3.0 (3–3) [1] 67.5 2.0 (0–68) [77]

66.7 2.0 (0–63) [15] 20.0 0 (0–2) [15] 40.0 0 (0–4) [15] 53.3 5.0 (0–36) [15] 87.5 1.5 (0–6) [16] 63.2 1.0 (0–95) [19] 55.8 1.0 (0–95) [95]

revealed unreadable chromatograms with double peaks. To check whether this was due to heterogeneous PCR products, we analysed them using the SSCP method as previously described [21]. Briefly, electrophoresis of six PCR products mixed with formamide was performed in a 9% native polyacrylamide gel at 8 °C during eight hours. The gel was stained with ethidium bromide and visualized on a UV transilluminator. To examine heterogeneity of amplified COI products within an individual P. laevis, the TA cloning procedure in the TOPO TA Cloning Kit (Invitrogen) was performed according to the manufacturer's instructions. Randomly chosen clones were used for plasmid DNA isolation (PureLink Quick Plasmid Miniprep Kit, Invitrogen) and subsequent sequencing. In total, 54 COI clones of P. laevis from Sava River fish were analysed by sequencing, including two clones from each of two worms and 25 clones from each of two other worms. In addition, 21 COI clones of P. laevis from the Sutla River fish (seven clones from each of three worms) were sequenced. Further amplification of COI sequences used for genetic analysis was performed by RT-PCR, to avoid amplification of COI pseudogenes. Total RNA was extracted from a single acanthocephalan using TRI reagent (MRC). Then cDNA was amplified using oligo dT primers and the Enhanced Avian HS RT-PCR Kit (Sigma-Aldrich) according to the manufacturer's instructions. PCR reactions using cDNA templates were performed using universal COI primers as described above, and amplicons were sequenced commercially (Macrogen, Netherlands). 2.2.2. Nuclear markers Two other nuclear markers were used for the molecular analysis of P. laevis: 18S rRNA and ITS1-5.8S rRNA-ITS2. Total DNA was extracted from a single acanthocephalan using a DNA purification kit (DNeasy Blood and Tissue Kit, Qiagen) according to the manufacturer's instructions. Primers and reaction conditions used to amplify 18S rRNA and ITS regions were described previously [1]. 2.2.3. Sequence analysis Paired forward and reverse reads of PCR products were obtained to ensure final sequence quality. All sequences obtained in this study have been deposited in GenBank under the following accession numbers:

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KF559284–KF559300 for the COI gene; KF559301–KF559304 and KJ819957–KJ820006 for mtDNA-like sequences; KF559305–KF559308 for the ITS region and KF559308 for the 18SrDNA gene. Sequence data were analysed using the GenBank BLAST program and ClustalX [22]. To investigate whether mtDNA-like sequences are results of one or several independent nuclear integration events, codon position bias was tested using the chi-squared test (χ2). If significant codon position bias is observed between two pseudogenes, they are assumed to have separate, selectively constrained mitochondrial origins [23]. The pairwise differences between 15 different mtDNA-like sequences with indels and/or stop codons were counted separately using MEGA 5 [24]. For each pairwise comparison, the χ2 test was performed to check whether differences between mtDNA-like sequences were equally distributed among the first, second and third codon positions (1:1:1) (df = 2, P b 0.05). A Bonferroni correction for multiple tests was applied for P (106 comparisons). To determine whether there are separate genetic clusters between different COI haplotypes from the Sava River basin and other isolates from continental Europe (accession numbers AY423348–AY423350; EF051062–EF051071), a haplotype network was constructed based on statistical parsimony. The maximum number of steps parsimoniously connecting two haplotypes was inferred by TCS v. 1.21, which estimates relationships among sequences [25]. A phylogenetic tree was constructed to analyse the relationships of COI haplotypes and mtDNA-like sequences obtained in our study with other Pomphorhynchus haplotypes from continental Europe, including P. laevis sequences under the accession numbers given above and P. tereticollis sequences under accession numbers AY423351–AY423353 and JN695504–JN695508. During this process, Bayesian inference using MrBayes v3.1.2 [26] was applied, as well as maximum parsimony and maximum likelihood using MEGA 5 [24]. A best-fit model of nucleotide substitution was selected using the Akaike information criterion (AIC) and Bayesian information criterion (BIC) in MEGA 5 [24]. The following parameters were used: invertebrate mitochondrial code, data partitioning by codon positions (Nst = 2 for all partitions), a Dirichlet prior, no covariation, four states with frequencies of a Dirichlet prior and gammadistributed rate variation across sites. The search was run with four chains for 2 million generations, with sampling every 100 generations and 5000 trees discarded as burning. A 50% majority rule consensus tree for each analysis was reconstructed using TreeGraph [27]. DNAsp 5 [28] and Arlequin 3.5 [29] were used to analyse intra- and interpopulation genetic diversity. Analysis of Molecular Variance (AMOVA) implemented in Arlequin was used to test the subdivision of the Sava River basin P. laevis population from the continental Europe population. Statistical significance (p) of the percentage of variation and fixation index (Fst) between populations was calculated.

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3. Results 3.1. Molecular analyses

2.3. Statistical analyses of the dynamics of P. laevis infection

3.1.1. A COI marker Before characterising P. laevis based on the COI gene, we searched for mtDNA-like sequences. SSCP analysis revealed heterogeneous COI amplicons from P. laevis individuals from the Sava River (Fig. 1). Four to six bands were preferentially amplified in a standard PCR reaction using universal primers, indicating mitochondrial-like sequences (Fig. 1). Sequence comparison of 54 COI clones from four P. laevis individuals from the Sava River (accession nos. KF559301–KF559304 and KJ819957–KJ820006) indicated an uncorrected divergence ranging from 0 to 20.1%, similar to the range of divergence (0–19.6%) between sequences of COI clones within an individual worm. An 85-nucleotide deletion was observed in four mtDNA-like sequences (KJ819964, KJ819966, KJ819975 and KJ819979), while other deletions ranged from one to three nucleotides. Only two singlenucleotide insertions were evident. 193 variable sites were found with 156 parsimony informative and 37 singletons. A shift in the reading frame and stop codons in all 54 sequences caused the protein product to be non-functional. There is no significant codon position bias in mtDNA-like sequences (χ2 test: df = 2, P = 0.2), suggesting that these mtDNA-like sequences descended from a unique mitochondrial transposition. However, sequence analysis of 21 COI clones from three P. laevis individuals from the Sutla River (accession nos. KF559286, KF559290 and KF559291) showed no evidence of genetic divergence, indicating the absence of mtDNA-like sequences in these specimens. All sequences amplified in RT-PCR lacked abnormalities (frameshifts or stop codons) and so they corresponded to functional COI genes and were used in subsequent genetic characterisation of P. laevis in order to avoid false amplification. The analysis of 24 COI sequences with a length of 647 bp (10 from the Sava River and 14 from the Sutla River) revealed 15 polymorphic sites, of which 11 corresponded to transitions (five parsimony informative sites) and four to transversion (no parsimony informative site). Fourteen haplotypes were observed (H = 14), 11 with one recording, one with two recordings, one with four recordings and one with seven recordings. Haplotype diversity (Hd) was 0.899 ± 0.048 and nucleotide diversity (π) was 0.00319 ± 0.00047. The haplotype network produced using statistical parsimony with a 95% connection limit showed distant genetic clusters of P. laevis strains from the Sava River basin in Croatia and other strains from continental Europe (Hungary, France, Czech Republic) based on 300-bp COI sequences (Fig. 2). The haplogroup representing P. laevis from the Sava River basin is separated from the other continental European P. laevis specimens by seven substitutions and six missing haplotypes (Fig. 2). The existence of population subdivision was further supported by AMOVA, which indicated significant divergence between the Croatian

For statistical analyses and graphical presentation of results, we used SigmaPlot for Windows 11.0. For the Sava River, statistical analyses of dynamics of P. laevis infection were performed only for the spring and autumn of 2006, because for both sampling campaigns in 2005 only the prevalence of P. laevis infection was determined. For the Sutla River, statistical analyses of dynamics of P. laevis infection were performed for both autumn periods, 2008 and 2009. Differences in abundance between two sampling periods were assessed using the Mann–Whitney rank sum test, whereas differences between sampling sites and between length categories were assessed using Kruskal–Wallis one-way analysis of variance on ranks, followed by subsequent pairwise comparisons using Dunn's test. The level of significance for each Mann–Whitney and Kruskal–Wallis test is indicated in the respective figures, while it was set at p b 0.05 for Dunn's test. Since the data were not normally distributed, we used nonparametric statistical analyses.

Fig. 1. Single-stranded conformation polymorphism analysis of six cloned fragments of the Pomphorhynchus laevis COI-like gene. Arrows point to multiple bands; M is 20-kb molecular marker.

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Fig. 2. Haplotype network for Pomphorhynchus laevis using statistical parsimony. Circles represent haplotypes (H), with circle size corresponding to haplotype frequency (number in parenthesis). Colours represent geographical origin. Small open circles indicate missing haplotypes. The position of each mutation is given in italics, relative to haplotypes.

Sava River basin population and the population that included other strains from continental Europe (Fst = 0.875, p ≤ 0.0001). Phylogenetic analysis (Fig. 3) confirmed the separate grouping of P. laevis from the Sava and Sutla Rivers relative to other European specimens of P. laevis and P. tereticollis in GenBank.

3.1.2. Nuclear markers The partial 18S rRNA sequence (1662 bp) was analysed from 20 P. laevis specimens from the Sava River (n = 10) and the Sutla River (n = 10). All sequences were identical and showed 100% identity to the 18S rRNA sequence of P. laevis under the GenBank accession no. AY423346 (country: France; host: intermediate — Gammarus pulex [1]). Sequencing of a partial ITS1-5.8S rDNA-partial ITS2 region (616 bp) in 25 P. laevis individuals (12 from the Sava River and 13 from the Sutla River) showed high percent similarity between individuals (99.6– 100%), except for one haplotype that showed 4.1% divergence. The predicted length of 5.8S rRNA was 94 nt (corresponding to 258–342 nt of DNA sequence), as reported for the genus Pomphorhynchus (accession nos. AY424669 [1], AY135415 [11] and JF706705 [10]). Sequence analysis of ITS1 (258 bp) revealed seven polymorphic sites, six of which corresponded to transitions and one to transversion (parsimony informative). Three haplotypes were observed (H = 3), one (H1) with 22 recordings, one (H2) with two recordings and one (H3) with one recording. Haplotype diversity (Hd) was 0.227 ± 0.106 and nucleotide diversity (π) was 0.00246 ± 0.00170. H1 and H2 haplotypes showed 100% and 99.6% similarity to the sequence under GenBank accession no. AY135415 (Czech Republic, S. cephalus [11]), while the H3 haplotype showed 100% similarity to the sequence under the accession no. AY135414 (Slovakia, S. cephalus [11]). Sequence analysis of ITS2 (272 bp) revealed 20 polymorphic sites, 10 of which corresponded to transitions and 10 to transversion (none parsimony informative). Three haplotypes were observed (H = 3), one (H1) with 18 recordings, one (H2) with six recordings and one (H3) with one recording. Haplotype diversity (Hd) was 0.080 ± 0.072 and nucleotide diversity (π) was 0.00588 ± 0.00531. These haplotypes showed the same similarity to GenBank sequences as the abovementioned ITS1 regions.

3.2. Dynamics of P. laevis infection The dynamics of P. laevis infection in European chub (S. cephalus) from the Sava and Sutla Rivers were analysed with the respect to their spatial and seasonal distribution, as well as their association with host length, condition and sex. Analyses in the Sava River were carried out on 139 chub caught at five sites in two different seasons, 80 in the spring and 59 in the autumn period of 2006. The co-infection of two acanthocephalan species, P. laevis and Acanthocephalus anguillae, was observed in the chub intestine at all locations in both samplings. Among detected acanthocephalans, 60.5% was identified as P. laevis in the spring, and as much as 86.1% in the autumn period. The prevalence and abundance of P. laevis infection were comparable in both seasons; prevalence was 52.5% in the spring and 50.8% in the autumn (Table 1). Prevalence decreased towards the downstream sites (Table 1). Analysis of spatial distribution of abundance revealed a similar pattern, with the lowest values observed at sites 4 and 5, but the differences were statistically significant only in the spring period (Fig. 4a, b). Analyses in the Sutla River were carried out on 172 chub caught at six sites in two autumn periods, 77 in the autumn of 2008 and 95 in the autumn of 2009. Co-infection with P. laevis and A. anguillae was found in only one chub at site 1 of the Sutla River during the first sampling period. Prevalence of P. laevis infection was 67.5% in the first sampling period, and somewhat lower in the second (55.8%), whereas abundance of parasitic infection was comparable in two sampling periods (Table 2). Spatial analysis of prevalence and abundance for the sampling campaign of 2009 was previously published [30]. Both prevalence and abundance were lowest at sampling site 2, confirming the results obtained during the previous sampling campaign of 2008 (Fig. 5). An association between fish length and abundance of parasitic infection was observed at both rivers. In chub from the Sava River, a slight trend towards greater abundance in bigger and therefore presumably older fish was observed, especially in autumn, but observed differences were not statistically significant (Fig. 6a, b). In chub from the Sutla River, statistically significant differences in numbers of parasites per fish in different size categories were observed in both sampling periods, with the highest abundance always obtained in chub longer than 25.0 cm

Fig. 3. Phylogenetic analysis of Pomphorhynchus laevis inferred from partial COI sequences (610 bp). Bootstrap values (1000 replicates) supported by Bayesian inference are given above, while values supported by maximum likelihood (N 65%) and maximum parsimony (N 65%) are given below.

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Fig. 5. The spatial distribution of abundance of Pomphorhynchus laevis infections in chub (Squalius cephalus) caught in the Sutla River in autumn of 2008. The results are presented as boxplots with boundaries indicating the 25th and 75th percentiles. The line within the box marks the median value; the whiskers above and below the box, the 10th and 90th percentiles; and the dots, the 5th and 95th percentiles.

Fig. 4. The spatial distribution of abundance of Pomphorhynchus laevis infections in chub (Squalius cephalus) caught in the Sava River in the spring (a) and autumn (b) of 2006. The results are presented as boxplots with boundaries indicating the 25th and 75th percentiles. The line within the box marks the median value; the whiskers above and below the box, the 10th and 90th percentiles; and the dots, the 5th and 95th percentiles.

(Fig. 6c, d). Clear evidence of an association of parasitic abundance with fish condition index or sex was not observed in samples from either the Sava or Sutla Rivers. Although at upstream locations of the Sava River, the intensity of P. laevis infection was greater in female chub than in male chub during the autumn of 2006 [31], the differences in parasite abundance between male and female chub at both rivers were not statistically significant during any of the sampling campaigns. 4. Discussion 4.1. Molecular analyses 4.1.1. A COI marker The discovery of cryptic speciation and strain formation of P. laevis in populations from continental Europe (Slovak Republic, Czech Republic, Hungary, France, Italy) [1,11,10] and the British Isles [9] motivated the present investigation into the genetic variability of P. laevis populations in the Sava River basin in Croatia. The Sava River is a tributary of the Danube River and therefore belongs to the Black Sea drainage basin. It is geographically distinct from the European rivers and lakes previously included in studies of P. laevis diversity. To gain insights into possible strain formation and ancestral lineage of P. laevis in continental Europe, we focused on S. cephalus as the parasite's definitive host, because it is believed to be the ancient, original host of P. laevis [3]. To ensure more reliable results, we used three gene regions to analyse genetic variability in P. laevis, including the COI gene. COI sequences have previously been used to analyse acanthocephalan populations [1, 10,12], cryptic speciation [10,32] as well as the taxonomic position of the acanthocephalan genera within families [21,33–35]. A single region of approximately 700 bp in the COI gene was used as a standard, universal marker to reconstruct phylogeny and establish “barcodes” for species. Conserved, universal primers for amplifying this region from different species were previously published; one of them is the Folmer region, designed for DNA barcoding of the broad range of metazoan invertebrates [20,36]. The primers used in our study have also been used in three earlier

studies of P. laevis genetic characterisation [1,10,13]. When we used PCR to amplify part of the COI gene in four specimens, we obtained products that were heterogeneous in both size and base composition. Despite the haploid nature of mtDNA, non-identical mtDNA sequences may exist in one individual; others have argued that it is better to use the term “mtDNA-like sequences” than “mtDNA pseudogenes” or “nuclear mitochondrial DNA (Numts)” to avoid a misnomer [37]. There are several instances in which unusual sequences can correspond to real mtDNA sequences rather than Numts: male and female mtDNA lineages within a species, aging mitochondrial genome, amplification of damaged DNA templates or the occurrence of other mitochondrial novelties (recombination, heteroplasmy through introgression, gene rearrangements and unconventional mtDNA architecture) [37]. Pseudogenes (Numts) of the COI gene were previously identified in the genus Acanthocephalus using these same universal PCR primers [19], but the Numts did not show a uniform distribution within this genus, since COI pseudogenes were not found in two species (A. anguillae and Acanthocephalus clavula). In our study, three of four mtDNA-like haplotypes clustered independently of the functional COI sequences. Brabec et al. [38] found no clustering of Numt haplotypes of caryophyllidean cestodes (nad3 + COI region) separately from the functional genes in phylogenetic analyses, while Benesh et al. [19] showed a separate clustering of Numts and functional COI sequences, indicating that the transfer and incorporation of mtDNA into the chromosome was an ancient, clade-specific event in Acanthocephalus evolution. Interestingly, previous studies of P. laevis COI sequences amplified from total DNA using the same Folmer primers as those in the present work did not detect mtDNA-like sequences [13]. One possible explanation is that the individuals from their study and ours, although of the same species, act as different strains with unique genetic histories. This possibility was also proposed for Acanthocephalus lucii [19] to explain the fact that specimens from England did not show the presence of COI pseudogenes, whereas specimens from other geographical locations did. Regardless of the explanation, our results highlight the need to exercise caution when using universal primers for P. laevis COI amplification. The haplotype network based on partial P. laevis COI sequences clearly showed genetic clustering of the specimens from the Sava River basin in Croatia with the specimens from continental Europe (Hungary, France, Czech Republic), which was confirmed by AMOVA (p ≤ 0.0001) (isolates of P. laevis from the British Islands [12] were excluded from the analysis since they were amplified with a different set of primers and did not overlap with our sequences.) However, the 300-bp COI sequences showed differences of 2.3%–3.3%, higher than the 2.2% divergence in a 295-bp COI sequence that led to the identification of two P. laevis strains that differed in definitive host species but not

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Fig. 6. The abundance of Pomphorhynchus laevis infections in different size categories of chub (Squalius cephalus) caught in the Sava River in the spring (a) and autumn (b) of 2006 and in the Sutla River in autumn periods of 2008 (c) and 2009 (d). The fish were classified in four categories according to total length (category 1: ≤15.0 cm; category 2: N15.0 cm to ≤ 20.0 cm; category 3: N20.0 cm to ≤25.0 cm; and category 4: N25.0 cm). The results are presented as in Fig. 4. Differences between sites are indicated with different letters (a, b) based on the Kruskal–Wallis one-way analysis of variance on ranks, followed by the post-hoc Dunn's test.

in geographical distribution [12]. In addition, P. laevis from the Sava River basin constitutes a subgroup in the P. laevis clade (with 99% bootstrap support), while other isolates in GenBank constitute another subgroup in the phylogenetic analysis, regardless of their definitive host or geographical location (Fig. 3). Therefore, our analysis of partial COI gene sequences strongly suggests that P. laevis from the Sava River basin in Croatia is a new strain of P. laevis in continental Europe. Differences in the use of intermediate hosts may also help explain parasite strain formation. It is possible that the Croatian strain of P. laevis uses Gammarus fossarum as an intermediate host, the distribution of which within the analysed part of the Sava River has been described [39]. This species is different from the Gammarus balcanicus, Echinogammarus stammeri and Gammarus roeseli, which have been described as intermediate hosts for other P. laevis populations in continental Europe [11].

4.1.2. Nuclear markers Analysis of ITS1 and ITS2 regions was less informative about strain formation because only three haplotypes were observed among 25 analysed sequences. These sequences showed high nucleotide identity (99.6–100%) with the isolates obtained from GenBank. In this way, our results confirm a previous report that ITS1/ITS2 sequences show less variation than COI sequences in P. laevis complex species [1]. We concur with the authors of that study that the ITS region should be used as a marker to study phylogenetic relationships among Pomphorhynchus species, while COI is a suitable marker for comparative analysis of intraspecific variation in different P. laevis forms at various geographical scales [1]. Our analysis of 18S rDNA sequences confirmed conservation of this region within P. laevis specimens, as all analysed sequences in our study were of the same haplotype. Nevertheless, this marker was suitable for elucidating evolutionary relationships among acanthocephalan

taxa [21,40,41], as well as for differentiating P. laevis sensu stricto and P. tereticollis [1]. 4.2. The dynamics of P. laevis infection P. laevis was the dominant intestinal parasite of S. cephalus in both the Sava and Sutla Rivers. The spatial distribution of P. laevis in both rivers was associated with differences in water quality at the selected sampling sites. Thus, the lower abundance of P. laevis at specific sampling sites (sites 4 and 5 on the Sava River, and site 2 on the Sutla River) may reflect the inferior water quality at those sites. For example, sites 4 and 5 on the Sava River (Lukavec Posavski and Jasenovac) showed slightly increased concentrations of several metals in fish tissues [31, 42–44], faecal water contamination [15], and increased water toxicity and moderate organic pollution [45,46]. Similarly, site 2 on the Sutla River showed increased concentrations of several dissolved metal species, as well as increased faecal water contamination [47]. Such ecosystem characteristics probably affected first-level intermediate hosts, which are known to be quite sensitive to pollution [48–50], resulting in lower abundance and prevalence of P. laevis infection. We observed a positive association between the abundance of parasite infection and fish size. This may be explained by the fact that larger/ older fish can feed on larger amphipods and consequently accumulate more parasites [51]. 5. Conclusions This study represents the first report of COI-like sequences in P. laevis species and also the first report of P. laevis strain formation in the Sava River basin. Clear taxonomic status of analysed P. laevis is important not just for further phylogeographic studies of the P. laevis complex, but also for water contamination studies. The P. laevis strain described here has

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been proposed as a sensitive biological indicator of metal bioavailability in river water, based on a study in the Sava River [31]. Acknowledgements We thank Dr. sc. Zrinka Dragun for her assistance with the statistical analysis. This work was supported by the Ministry of Science, Education and Sport of the Republic of Croatia (project no. 098-0982934-2752) and the European Union FP6 SARIB Project (INCO-CT-2004-509160). References [1] Perrot-Minnot M-J. Larval morphology, genetic divergence, and contrasting levels of host manipulation between forms of Pomphorhynchus laevis (Acanthocephala). Int J Parasitol 2004;34:45–54. [2] Kennedy CR. Ecology of the Acanthocephala. Cambridge, UK: Cambridge University Press; 2006. [3] Kennedy CR, Broughton PF, Hine PM. The status of brown and rainbow trout, Salmo trutta and Salmo gairdneri as hosts of the acanthocephalan Pomphorhynchus laevis. J Fish Biol 1978;13:265–75. [4] Kennedy CR. The status of flounders, Platichthys flesus L., as hosts of the acanthocephalan Pomphorhynchus laevis (Müller) and its survival in marine conditions. J Fish Biol 1984; 24:135–49. [5] Brown AF. Anatomical variability and secondary sexual characteristics in Pomphorhynchus laevis (Müller, 1776) (Acanthocephala). Syst Parasitol 1987; 9:213–9. [6] Munro MA, Whitfield PJ, Diffley R. Pomphorhynchus laevis (Müller) in the flounder, Platichthys flesus L., in the tidal Thames: population structure, microhabitat utilization and reproductive status in the field and under conditions of controlled salinity. J Fish Biol 1989;35:719–35. [7] Dudiňák V, Šnábel V. Comparative analysis of Slovak and Czech populations of Pomphorhynchus laevis (Acanthocephala) using morphological and isoenzyme analyses. Acta Zool Univ Comen 2001;44:47–56. [8] Guillén-Hernández S, Whitfield PJ. A comparison of freshwater and marine/estuarine strains of Pomphorhynchus laevis occurring sympatrically in flounder, Platichthys flesus, in the tidal Thames. J Helminthol 2001;75:237–43. [9] O'Mahony EM, Kennedy CR, Holland CV. Comparison of morphological characters in Irish and English populations of the acanthocephalan Pomphorhynchus laevis (Müller, 1776). Syst Parasitol 2004;59:147–57. [10] Špakulová M, Perrot-Minnot MJ, Neuhaus B. Resurrection of Pomphorhynchus tereticollis (Rudolphi, 1809) (Acanthocephala: Pomphorhynchidae) based on new morphological and molecular data. Helminthologia 2011;48:268–77. [11] Král'ová-Hromadová I, Tietz DF, Shinn AP, Špakulová M. ITS rDNA sequences of Pomphorhynchus laevis (Zoega in Müller, 1776) and P. lucyi William & Rogers, 1984 (Acanthocephala: Palaeacanthocephala). Syst Parasitol 2003;56:141–5. [12] O'Mahony EM, Bradley DG, Kennedy CR, Holland CV. Evidence for the hypothesis of strain formation in Pomphorhynchus laevis (Acanthocephala): an investigation using mitochondrial DNA sequences. Parasitology 2004;129:341–7. [13] Moret Y, Bollache L, Wattier R, Rigaud T. Is the host or the parasite the most locally adapted in an amphipod–acanthocephalan relationship? A case study in a biological invasion context. Int J Parasitol 2007;37:637–44. [14] Kennedy CR, Bates RM, Brown AF. Discontinuos distribution of the fish acanthocephalans Pomphorhynchus laevis and Acanthocephalus anguillae in Britain and Ireland: an hypothesis. J Fish Biol 1989;34:607–19. [15] Kapetanović D, Dragun Z, Valić D, Teskeredžić Z, Teskeredžić E. Enumeration of heterotrophs in river water with spread plate method: comparison of yeast extract agar and R2A agar. Fresenius Environ Bull 2009;18:1276–80. [16] Bush AO, Lafferty KD, Lotz JM, Shostak AW. Parasitology meets ecology on its own terms: Margolis et al. revisited. J Parasitol 1997;83:575–83. [17] Nash RDM, Valencia AH, Geffen AJ. The origin of Fulton's condition factor — setting the record straight. Fisheries 2006;31:236–8. [18] Moravec F. Metazoan parasites of salmonid fishes of Europe. Prague: Academia; 2004. [19] Benesh DP, Hasu T, Soumalainen LR, Valtonen ET, Tiirola M. Reliability of mitochondrial DNA in an acanthocephalan: the problem of pseudogenes. Int J Parasitol 2006;36: 247–54. [20] Folmer O, Black M, Hoen W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 1994;3:294–9. [21] Vardić Smrzlić I, Valić D, Kapetanović D, Dragun Z, Gjurčević E, Ćetković H, et al. Molecular characterisation and infection dynamics of Dentitruncus truttae from trout (Salmo trutta and Oncorhynchus mykiss) in Krka River, Croatia. Vet Parasitol 2013;197(3–4):604–13. http://dx.doi.org/10.1016/j.vetpar.2013.07.014. [22] Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res 1997;25:4876–82. [23] Bensasson D, Zhang D-X, Hewitt GM. Frequent assimilation of mitochondrial DNA by grasshopper nuclear genomes. Mol Biol Evol 2000;17:406–15.

[24] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2001;28:2731–9. [25] Clement M, Posada D, Crandall KA. TCS: a computer program to estimate gene genealogies. Mol Ecol 2000;9:1657–9. [26] Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 2001;17:754–5. [27] Stöver BC, Müller KF. TreeGraph 2: combining and visualizing evidence from different phylogenetic analyses. BMC Bioinformatics 2010;11:7. [28] Librado P, Rozas J. DnaSP ver. 5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 2009;25:1451–2. [29] Excoffier L, Lischer HEL. Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 2010;10: 564–7. [30] Dragun Z, Filipović Marijić V, Kapetanović D, Valić D, Vardić Smrzlić I, Krasnići N, et al. Assessment of general condition of fish inhabiting a moderately contaminated aquatic environment. Environ Sci Pollut Res 2013;20:4954–68. [31] Filipović Marijić V, Vardić Smrzlić I, Raspor B. Effect of acanthocephalan infection on metal, total protein and metallothionein concentrations in European chub from a Sava River section with low metal contamination. Sci Total Environ 2013; 463–464:772–80. [32] Steinauer ML, Nickol BB, Ortí G. Cryptic speciation and patterns of phenotypic variation of a highly variable acanthocephalan parasite. Mol Ecol 2007;16:4097–109. [33] Garciá-Varela M, Pérez-Ponce de León G. Validating the systematic position of Profilicollis Meyer, 1931 and Hexaglandula Petrochenko, 1950 (Acanthocephala: Polymorphidae) using cytochrome C oxidase (cox 1). J Parasitol 2008;94:212–7. [34] Garciá-Varela M, Pérez-Ponce de León G, Aznar FJ, Nadler SA. Systematic position of Pseudocorynosoma and Andracantha (Acanthocephala, Polymorphidae) based on nuclear and mitochondrial gene sequences. J Parasitol 2009;95:178–85. [35] Garciá-Varela M, Pérez-Ponce de León G, Aznar FJ, Nadler SA. Erection of Ibirhynchus gen.nov. (Acanthocephala: Polymorphidae) based on molecular and morphological data. J Parasitol 2011;97:97–105. [36] Herbert PDN, Cywinska AS, Ball L, deWaard JR. Biological identifications through DNA barcodes. Proc R Soc Lond B Biol Sci 2003;270:313–21. [37] Schizas NV. Misconceptions regarding nuclear mitochondrial pseudogenes (Numts) may obscure detections of mitochondrial evolutionary novelties. Aquat Biol 2012; 17:91–6. [38] Brabec J, Scholz T, Králová-Hromadová I, Bazsalovicsová E, Olson PD. Substitution saturation and nuclear paralogs of commonly employed phylogenetic markers in the Caryophyllidea, an unusual group of non-segmented tapeworms (Platyhelminthes). Int J Parasitol 2012;42:259–67. [39] Garašić D, Meštrov M. Utjecaj zagađenja na distribuciju i dinamiku populacija vrste Gammarus fossarum Koch, 1836. u rijeci Savi (The influence of pollution on the distribution and population dynamics of the species Gammarus fossarum Koch, 1836. in the Sava River). Acta Biol 1983;48:87–96 [abstract in english]. [40] Garciá-Varela M, Nadler SA. Phylogenetic relationships of Palaeacanthocephala (Acanthocephala) inferred from SSU and LSU rDNA gene sequences. J Parasitol 2005;91:1401–9. [41] Verweyen L, Klimpel S, Palm HW. Molecular phylogeny of the Acanthocephala (Class Palaeacanthocephala) with a paraphyletic assemblage of the orders Polymorphida and Echinorhynchida. PLoS One 2011;6:e28285. [42] Dragun Z, Podrug M, Raspor B. Combined use of bioindicators and passive samplers for the assessment of river water contamination with metals. Arch Environ Contam Toxicol 2009;57:211–20. [43] Podrug M, Raspor B, Erk M, Dragun Z. Protein and metal concentrations in two fractions of hepatic cytosol of the European chub (Squalius cephalus L.). Chemosphere 2009;75:843–9. [44] Filipović Marijić V, Raspor B. Site-specific gastrointestinal metal variability in relation to the gut content and fish age of indigenous European chub from the Sava River. Water Air Soil Pollut 2012;223:4769–83. [45] Krča S, Žaja R, Čalić V, Terzić S, Grubešić MS, Ahel M, et al. Hepatic biomarker responses to organic contaminants in feral chub (Leuciscus cephalus) — laboratory characterization and field study in the Sava river, Croatia. Environ Toxicol Chem 2007;26(12):2620–33. [46] Källqvist T, Milačić R, Smital T, Thomas KV, Vranes S, Tollefsen KE. Chronic toxicity of the Sava River (SE Europe) sediments and river water to the algae Pseudokirchneriella subcapitata. Water Res 2008;42:2146–56. [47] Dragun Z, Kapetanović D, Raspor B, Teskeredžić E. Water quality of medium size watercourse under baseflow conditions: the case study of river Sutla in Croatia. Ambio J Hum Environ 2011;40:391–407. [48] Galli P, Crosa G, Ambrogi AO. Heavy Metals concentrations in acanthocephalans parasites compared to their fish host. Chemosphere 1998;37(14–15):2983–8. [49] Galli P, Crosa G, Mariniello L, Ortis M, D'Amelio S. Water quality as a determinant of the composition of fish parasite communities. Hydrobiologia 2001;452:173–9. [50] Kennedy CR. Freshwater fish parasites and environmental quality: an overview and caution. Parassitologia 1997;39:249–54. [51] Emde S, Rueckert S, Palm HW, Klimpel S. Invasive Ponto-Caspian amphipods and fish increase the distribution range of the acanthocephalan pomphorhynchus tereticollis in the river rhine. PLoS One 2012;7(12):e53218. http://dx.doi.org/10. 1371/journal.pone.0053218.