Ecologica Montenegrina 39: 69-75 (2021) This journal is available online at: www.biotaxa.org/em http://dx.doi.org/10.37828/em.2021.39.7 Molecular data on Phyllodistomum macrocotyle (Digenea: Gorgoderidae) from an intermediate host Dreissena polymorpha (Bivalvia: Dreissenidae) in the Northern Dvina River Basin, Northwest Russia OKSANA V. TRAVINA, YULIA V. BESPALAYA*, ALEXANDER V. KONDAKOV, OLGA V. AKSENOVA, IRINA S. KHREBTOVA & ALEXANDER V. KROPOTIN N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Severnaya Dvina Emb. 23, 163000 Arkhangelsk, the Russian Federation *Corresponding author. E-mail: [email protected] Received 12 December 2020 │ Accepted by V. Pešić: 30 January 2021 │ Published online 8 February 2021. Abstract We report the first molecular identification of Phyllodistomum macrocotyle (Digenea: Gorgoderidae) from the Northern Dvina River Basin, Northwest Russia. Comparative analysis of our sequence was 100% identical to the 28S and ITS2 rDNA sequences of the species P. macrocotyle in zebra mussels discovered in Poland (Wigry Lake), Lithuania (Elektrenai Reservoir and Sirvinta River), Russia, Belarus (Lepelskoe Lake and Lukomskoe Lake), and in fish Leuciscus idus, Scardinius erythrophthalmus in Russia (Rybinsk Reservoir on the Volga River). At the same time, some sequences of the 28S rDNA of P. macrocotyle in zebra mussel detected in Lithuania, and Belarus were different from our sequence on 0.2-0.6%. The ITS2 sequences which have 100% similarity with our specimens of P. macrocotyle were discovered in sporocyst of phyllodistomes from its intermediate host D. polymorpha in Poland, Lithuania, Russia, and Belarus. Our observation in the Yuras and Isakogorka rivers represents the northernmost record of this host-specific parasite, to the best of our knowledge. Key words: Phyllodistomum macrocotyle, Dreissena polymorpha, molecular identification, Northern Dvina River Basin, Northern European Russia. Introduction The trematode genus Phyllodistomum Braun, 1899 is probably the most diverse genus within the Digenea and contains around 120 species. This genus has a worldwide distribution, containing parasites of amphibians and both marine and freshwater fishes. Trematodes typically inhabit the urinary bladder and/or ureters of fishes (Pérez-Ponce de León et al. 2015; Petkevičiūtė et al. 2018, 2020). The taxonomy of the genus Phyllodistomum remains unresolved and taxonomic confusion is caused by the difficulties in identifying the species of the genus (Stunžėnas et al. 2017; Petkevičiūtė et al. 2018, 2020). The genus Ecologica Montenegrina, 39, 2021, 69-75 MOLECULAR DATA ON PHYLLODISTOMUM MACROCOTYLE Phyllodistomum needs a revision based on an integrative approach that includes both molecular analysis and detailed morphological descriptions (Peribáñez et al. 2011; Petkevičiūtė et al. 2020). The microcercous cercariae of Phyllodistomum develop in sporocysts localized in the gills of zebra mussel Dreissena polymorpha (Petkevičiūtė et al. 2015, 2020; Travina et al. 2019). Phyllodistomum macrocotyle (Lühe, 1909) is the only one valid species of Phyllodistomum for the zebra mussel (Petkevičiūtė et al. 2015, 2020; Stunžėnas et al. 2017). The Phyllodistomum species is found in European populations of D. polymorpha in the Netherlands, Poland, Belarus, Russia, Ukraine, Kazakhstan (Molloy et al. 1996, Tyutin et al. 2005), Lithuania (Stunžėnas et al. 2004; Petkevičiūtė et al. 2015) and Spain (Peribáñez et al. 2011). The infection of D. polymorpha with sporocysts of P. macrocotyle in the Northern Dvina River Basin, Arkhangelsk region, Northwest Russia) was detected in 2016 (Travina et al. 2019) and is a northernmost record of this host specific parasite. However, this record was based exclusively on the morphological identification of the collected specimens. To date, no sequences of this species are available from Northwest Russia, to the best of our knowledge. The ITS2 and 28S rDNA sequences of P. macrocotyle were obtained from sporocyst of phyllodistomes from its intermediate host D. polymorpha in Poland, Lithuania, Russia (the Rybinsk Reservoir on the Volga River) and Belarus (Stunžėnas et al. 2004; Petkevičiūtė et al. 2015, 2020), and also in Spain (Peribáñez et al. 2011). The ITS2 and 28S rDNA sequences of adult P. macrocotyle from several fish species were obtained in Russia (the Rybinsk Reservoir on the Volga River) from Leuciscus idus and Scardinius erythrophthalmus (Petkevičiūtė et al. 2020). The ITS2 sequences, identical to some sequences from the Belarus population were received in Spain from Cyprinus carpio, Rutilus rutilus and S. erythrophthalmus (Peribáñez et al. 2011). This study provides the molecular identification of the northernmost record of P. macrocotyle from the intermediate host Dreissena polymorpha (Arkhangelsk region, Northern European Russia) for the first time. Figure 1. Map of the study area: A) Geographic position of the research area (red color frame and red color point); B) The Northern Dvina River Basin (red color flags indicate points where zebra mussels infected with Phyllodistomum macrocotyle were found); C) Habitat of zebra mussel, the Yuras River; D) Trematode sporocysts located within the gills of Dreissena polymorpha. 70 TRAVINA ET AL. Materials and methods Samples of D. polymorpha were collected from the Yuras and Isakogorka rivers in the Northern Dvina River Basin (Arkhangelsk) in the summer periods (June-August) from 2016 to 2020 (Fig. 1). The samples of D. polymorpha were gathered using a rectangular hand net, a Petersen grab and also manual collection from various substrates, (for further information consult: Travina et al. 2019). The materials are stored in the collection of the Russian Museum of the Biodiversity Hotspots (RMBH) of the N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia. Trematodes were isolated from gills of D. polymorpha (Fig. 1D) and placed in 96% ethanol, then we used the detected trematodes for molecular genetic analysis. Total genomic DNA was extracted from free 96% ethanol-preserved samples of trematodes using the NucleoSpin Tissue Kit (Macherey-Nagel GmbH & Co. KG, Germany), following the manufacturer’s protocol. For molecular analyses, we obtained sequences of the two nuclear markers: internal transcribed spacer 2 (ITS2) and the 28S ribosomal DNA (28S rDNA), which are widely used as a genetic markers in such studies (Petkevičiūtė et al. 2015, Aksenova et al. 2016). The ITS2 and 28S rDNA sequences were amplified by polymerase chain reaction (PCR) using the following primers: 3S (Bowles et al. 1995) and ITS4 (White et al. 1996), C1 and D2 (Jovelin & Justine 2001), respectively. The PCR mix contained approximately 200 ng of total cell DNA, 10 pmol of each primer, 200 μmol of each dNTP, 2.5 μl of PCR buffer (with 10 × 2 mmol MgCl2), 0.8 units Taq DNA polymerase (SibEnzyme Ltd.), and H2O was added for a final volume of 25 μl. Thermocycling was implemented with markers-specific PCR programs as follows: (i) ITS2: 95 °C (4 min), followed by 32 cycles at 94 °C (50 sec), 54 °C (50 sec), 72 °C (50 sec) and a final extension at 72 °C (5 min); (ii) 28S rDNA: 95 °C (4 min), followed by 24 cycles at 94 °C (50 sec), 60 °C (50 sec), 72 °C (50 sec) and a final extension at 72 °C (5 min). Forward and reverse sequencing were performed on an automatic sequencer (ABI PRISM3730, Applied Biosystems) using the ABI PRISM BigDye Terminator version 3.1 reagent kit. The resulting sequences were checked using a sequence alignment editor BioEdit version 7.2.5 (Hall 1999). The nucleotide sequences of the sporocysts of trematodes were identified using the Basic Local Alignment Search Tool, BLAST (Johnson et al. 2008). In addition, 7 ITS2 sequences and 23 28S rDNA sequences were obtained from NCBI’s GenBank (Table 1). The ITS2 and 28S rDNA sequence datasets were aligned by using the MUSCLE algorithm of MEGA7 (Kumar, Stecher, & Tamura 2016). The 28S rDNA alignment was additionally processed through the GBlocks Server v. 0.91b (Talavera & Castresana 2007) to exclude hypervariable positions. The final length alignment of ITS2 was 447 bp and 28S rDNA alignment after GBlocks treatment was 1094 bp. The maximum likelihood 28S rDNA phylogenies (Fig. 2) were reconstructed using IQ-TREE version 1.6.11 (Nguyen et al. 2015) with an automatic detection of the best evolutionary model via an online resource of the Center for Integrative Bioinformatics Vienna, Austria (Trifinopoulos et al. 2016). The node support values were estimated using an ultrafast bootstrap algorithm (Hoang et al. 2018). Results Sporocysts of trematodes of the genus Phyllodistomum were found on the gills of D. polymorpha from the Yuras River and Isakogorka River during the study of the settlement of Dreissena in the watercourses of the Northern Dvina River Basin (Fig. 1). The present analysis includes 499 specimens of D. polymorpha of which 446 were collected from the Yuras River and used for a previous analysis of trematode infection in D. polymorpha (Travina et al. 2019). P. macrocotyle (Lühe, 1909) sporocyst infection was recorded in three individuals of D. polymorpha from the Yuras River and one individual of D. polymorpha from the Isakogorka River. The overall percentage of infected individuals of D. polymorpha by P. macrocotyle was <1% for each river. Phylogenetic analysis using the obtained nucleotide sequences of 28S (acc. no. MW362297, MW362298 and MW543441) with sequences taken from