Sarabeev_02_2022.indd UDC 595.122(438) MOLECULAR CHARACTERIZATION OF THE FIRST REPORTED NEOPLAGIOGLYPHE MEGASTOMUS (DIGENEA, OMPHALOMETRIDAE) IN POLAND V. Sarabeev1,2*, M. Ovcharenko2, A. S. Ahmed1, R. A. Sueiro3, J. M. Leiro3 1Department of Biology, Zaporizhzhia National University, Zhukovskogo 66, 69063 Zaporizhzhia, Ukraine 2Institute of Biology and Science of Earth, Pomeranian University in Słupsk, Arciszewskiego 22b, 76200 Słupsk, Poland 3Department of Microbiology and Parasitology, Institute of Research in Biological and Chemical Analysis,University of Santiago de Compostela, 15782 Santiago de Compostela, Spain *Corresponding author E-mail: vosa@ext.uv.es, volodimir.sarabeev@gmail.com V. Sarabeev (https://orcid.org/0000-0003-4724-3141) M. Ovcharenko (https://orcid.org/0000-0001-9208-8959) Molecular Characterization of the First Reported Neoplagioglyphe megastomus (Digenea, Omphalometridae) in Poland. Sarabeev, V., Ovcharenko, M., Ahmed, A., Sueiro, R. A., Leiro, J. M. — Th e diversity and taxonomy of metacercariae infecting freshwater amphipods of Poland are predominantly poorly known. During parasitological surveys of Gammarus pulex (Crustacea: Amphipoda) in the Pomeranian region of Poland in 2020 and 2021, some specimens of crustaceans were found to harbour metacercariae. Out of seven observed localities, metacercariae were found in the stream close to Krępa Słupska and the Lupawa River close to Smoldzino. Th ey were morphologically studied and sequenced using universal eukaryotic primers that amplify V4–V5 regions of 18S rRNA. Th e BLAST analysis and phylogenetic reconstructions aid to defi ne the affi liation of the revealed metacercariae within the Omphalometridae Looss, 1899. Th e further diff erential analysis based on morphologic information led to the conclusion that the metacercarial form studied herein represents Neoplagioglyphe megastomus (Baer, 1943). Th e degree of morphometric variations of taxonomic important features in populations from diff erent geographic regions was followed up. Th e reported here form was characterized by a larger body size that was aff ected by the processing methods used in the present and previous studies. To our knowledge, this is the fi rst report of Neoplagioglyphe megastomus in Poland and is the fi rst molecular characterization of the worm. K e y w o r d s : 18S rRNA, Gammarus pulex, Pomeranian region, metacercaria. Introduction Digeneans are a group of phylogenetically diverse parasitic fl atworms that have complex life cycles with two or three hosts, where vertebrates are defi nitive hosts and molluscs are fi rst intermediate hosts. Th e infective metacercarial stage may pass through a variety of invertebrate and vertebrate hosts serving as second intermediate hosts in which they undergo extensive organogenesis (Saville and Irwin, 1991). As crustaceans are highly abundant in the aquatic environment and play a key role as an important source of nutrition, digeneans are known to frequently include pelagic and benthic crustaceans in their life cycle to use the trophic interactions in food webs for facilitating the transmission of parasites to the successive hosts (Busch et al., 2012). Metacercariae diversity in amphipods was recently evaluated by Bojko and Ovcharenko (2019), who counted 14 and over 30 species of digeneans from marine and freshwater amphipods, respectively. Zoodiversity, 56(2):83–90, 2022 DOI 10.15407/zoo2022.02.083 Fauna and Systematics 84 V. Sarabeev, M. Ovcharenko, A. S. Ahmed, R. A. Sueiro, J. M. Leiro Since Digenea commonly includes species, genera and families with few morphological distinctions, their accurate identifi cation is complicated (Gibson, Jones and Bray, 2002; Goswami et al., 2013). Th e metacercarial stages oft en lack reliable distinguishing morphological characters, especially for structures associated with the reproductive system, and thus their identifi cation to the species level is complicated or even impossible. However morphological data remain the cornerstone of trematode systematics (Blasco-Costa et al., 2016). Over recent decades there has been an increased integration of genetic data to overcome problems in establishing accurate species limits and higher taxonomic groups of digeneans (Pérez-Ponce de León and Hernández-Mena, 2019). Th e current taxonomy and classifi cation of digeneans is based on molecular data predominantly derived from phylogenetic assessments of two nuclear rRNA genes, 18S and 28S (Olson et al., 2003; Blasco-Costa et al., 2016; Pérez-Ponce de León and Hernández-Mena, 2019). During parasitological surveys of Gammarus pulex (L.) (Crustacea: Amphipoda) in the Pomeranian region of Poland in 2020 and 2021, some specimens of crustaceans were found to harbour metacercariae. Th e metacercariae were studied alive, microphotographed and measured with subsequent fi xation and total genomic DNA extraction. Th e obtained sequences were analysed using the BLAST Sequence Analysis Tool and phylogenetic reconstruction of the closest taxa found in GenBank. Th e fi nal species identifi cation was performed using diagnostic morphological features. Material and methods M a t e r i a l c o l l e c t i o n a n d p r o c e s s i n g G. pulex were collected from streams and rivers in the Pomeranian region of Poland. In total 233 individuals from seven localities were surveyed for parasites (table 1). Amphipods were measured under a stereomicroscope (SMZ-161 with digital camera Moticam BTU) and dissected on the object-glass. Parasites were counted, excysted with syringe needles and transferred for further inspection under a compound microscope. Cysts and metacercariae were microphotographed and measured alive (table 2) using a digital camera Optikam B3 and microscope Delta Optical Evolution 300. Selected specimens were preserved in absolute ethanol for molecular analysis. Th e line drawing was prepared from a set of microphotographs obtained from alive worms with magnifi cations 100x and 400x. Similarly to previous studies (Blasco-Costa et al., 2006, 2010), ratios were counted to compare literature descriptions with specimens of digeneans collected here. Parasites were identifi ed to the species level based on morphology aft er defi ning their family affi nity with aid of molecular analysis. D N A e x t r a c t i o n , a m p l i f i c a t i o n , s e q u e n c i n g a n d a n a l y s i s Genomic DNA was extracted from a single metacercaria using a quick alkaline lysis protocol (Klimyuk et al., 1993; Stanton, McNicol and Steele, 1998). Individual metacercariae were transferred to 10 μL 0.25 M NaOH in 0.2 mL tube and sonicated for several seconds. Th e sample was incubated in NaOH for 3 min at 95 °C and subsequently neutralized by addition of 9 μL 0.25 M HCl and 8 μL 1 M Tris-HCl (pH 8.5), 1 μL 2 % Triton X-100 was also added as detergent. Th e mixture was again incubated for 3 min at 95 °C. Th e PCR was performed using universal eukaryotic primers F-566:5’-CAG CAG CCG CGG TAA TTC C-3’ and R-1200:5’-CCC GTG TTG AGT CAA ATT AAG C-3’ to amplify V4 and V5 variable regions of 18S rRNA gene as those with high taxonomic information (Hadziavdic et al., 2014). Th e PCR mixtures (25 μL) contained reaction buff er, 0.2 mM of each deoxynucleoside triphosphate (dNTPs, Nzytech, Portugal), 0.4 μM of each primer; 0.4 units of high fi delity NZYProof DNA polymerase (Nzytech) and 50 ng of genomic DNA. Th e reactions were run in an automatic thermocycler (T100TM Th er- mal Cycler, BioRad, USA) as follows: initial denaturing at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, annealing at 57 °C for 45 s, and 72 °C for 1 min; and fi nally, a 7 min extension phase at 72 °C. Th e PCR products were confi rmed by 1.5 % agarose gel in Tris-acetate ethylenediaminetetraacetic acid buff er containing Sybr Green at 1× concentration, to verify the presence of bands of the correct size under a variable-intensity 312 nm ultraviolet (UV) transilluminator (Spectroline, USA). Th e PCR product was purifi ed by a PCR purifi ca- T a b l e 1 . Results of Gammarus pulex samplings from streams and rivers in the Pomeranian region of Poland with information on the date, locality, number of studied crustaceans and infection parameters, prevalence (P) and mean abundance (A) with an intensity range in parentheses Sample ID Date Locality name Geographic coordinates n P, % A GPO 27/09/2020 Orzechowa 54.598841, 16.918841 43 0 0 GPLD 10/10/2020 Lesny Dwor 54.358391, 17.155713 28 0 0 GPSL 31/01/2021 Smoldzino Lupawa 54.662085, 17.212266 31 10 0.23 (1–4) GPDP 20/02/2021 Debki Piasnicy 54.832288, 18.061855 30 0 0 GPSS 28/02/2021 Slupsk Stadion 54.475260, 17.042841 30 0 0 GPKS 05/03/2021 Krępa Słupska 54.403371, 17.047010 41 38 0.8 (1–8) GPSW 25/03/2021 Wodnica Slupia 54.556568, 16.875233 30 0 0 85Molecular Characterization of the First Reported Neoplagioglyphe megastomus... tion kit (PureLinkTM, Invitrogen, USA) and was sequenced in complementary directions using Sanger sequenc- ing service (Eurofi ns Genomics, Germany). Obtained forward and reverse sequences were assembled and visualised using MEGA-11 (Tamura, Stech- er and Kumar, 2021). Th e obtained sequences were compared with GenBank entries by using BLAST tool (Altschul, 1997) to defi ne the taxonomic position of the metacercaria. According to the Blast search, 20 ad- T a b l e 2 . Comparative metrical data for metacercariae of Neoplagioglyphe megastomus from Gammarus pulex and adults from water shrews Metacercariae Adult Metrical data Gammarus pulex Neomys fodiens Neomys anomalus Mean Range n Range Range Range Body length 930.5 866–995 2 500–600 375–600 672–800 Maximum body width 230.5 220–241 2 120 200–300 208–210 Forebody length 415 352–478 2 – – – Oral sucker length 150.3 137–164 3 110–122 126–150 128–147 Oral sucker width 132.6 113–150 5 90–108 115–126 128 Prepharynx length 32.5 22–43 2 – – – Pharynx length 45.3 41–53 6 – 40–50 38–57 Pharynx width 50.8 35–65 4 – – 38–54 Ventral sucker length 66.5 56–77 6 47–58 65–73 67–96 Ventral sucker width 65 51–90 6 – – 60–96 Cirrus sac length 211 186–236 2 – 216–240 – Cirrus sac width 36 32–40 2 – 36–40 – Cirrus length 83 61–105 2 – – – Cirrus width 9 7–11 2 – – – Prostatic bulb length 22 – 1 – – – Prostatic bulb width 15 – 1 – – – Genital atrium length 19.5 17–22 2 – – – Genital atrium width 15.5 15–16 2 – – – Proximal portion of seminal vesicle length 23 – 1 – – – Proximal portion of seminal vesicle width 11 – 1 – – – Distal portion of seminal vesicle length 49 – 1 – – – Distal portion of seminal vesicle width 12 – 1 – – – Metraterm length 86.5 80–93 2 – – – Metraterm width 18.5 15–22 2 – – – Anterior testis length 90.5 78–109 4 – – 108–118 Anterior testis width 78.5 62–102 4 – – 92–93 Posterior testis length 94.8 71–124 4 – – 96–115 Posterior testis width 76.8 60–100 4 – – 89–115 Ovary length 53.8 42–62 4 – – 76–83 Ovary width 57.8 50–62 4 – – 51–67 Laurer’s canal length 105 1 – – – Laurer’s canal width 13 1 – – – Mehlis’ gland length 99 1 – – – Mehlis’ gland width 84 1 – – – Excretory bladder length 219 1 – – – Post-caecal fi eld length 77.5 67–88 2 – – – Post-testicular fi eld length 127 126–128 2 – – – Ratios* – – – Maximum body width as a percentage of body length 25 22–28 2 – 18–36 24–25 Length of the forebody as a percentage of body length 44 40–48 2 – 37–46 42–46 Sucker length ratio 2.0 1.9–2.1 3 – 2.1–2.6 1.4–1.5 Sucker width ratio 2.0 1.7–2.5 5 – 2.0–2.2 1.3–1.4 Cirrus sac length to ventral sucker length 2.8 2.6–3.1 2 – 2.4–4.1 1.9–2.1 Post-testicular fi eld length as a percentage of body length 13.7 13–15 2 – 14–20 15.1–15.3 Post-caecal fi eld length as a percentage of body length 8.4 7-10 2 – 3–8 5.0–5.5 Cysta length 315.3 273–352 4 520 – – Cysta width 249.3 205–280 4 360 – – References Th e present study Baer (1943) Baer (1943) Matskási (1971) * For literary material the ratios were evaluated from published line drawings. 86 V. Sarabeev, M. Ovcharenko, A. S. Ahmed, R. A. Sueiro, J. M. Leiro T a b l e 3 . Results of BLAST analysis performed on 18S rRNA sequence obtained from the Gammarus pulex metacercaria. Best hits (with highest similarity scores) are shown as retrieved in October 2021 from GenBank Species Family Superfamily Coverage, % Similarity, % GenBank Accession number Rubenstrema exasperatum Omphalometridae Plagiorchioidea 100 98.20 AJ287572.1 Mesocoelium lanfrediae Brachycoeliidae Plagiorchioidea 100 96.82 JQ886404.1 Brachycoelium salamandrae Brachycoeliidae Plagiorchioidea 100 96.54 AY222160.1 Opisthioglyphe ranae Telorchiidae Plagiorchioidea 100 96.54 AY222157.1 Choanocotyle nematoides Choanocotylidae Plagiorchioidea 100 96.40 EU196357.1 Auridistomum chelydrae Auridistomidae Plagiorchioidea 100 96.13 AY222159.1 Choanocotyle hobbsi Choanocotylidae Plagiorchioidea 100 95.99 MW682818.1 Choanocotyle platti Choanocotylidae Plagiorchioidea 100 95.99 EU196355.1 Telorchis assula Telorchiidae Plagiorchioidea 100 95.84 AY222156.1 Oschmarinella macrorchis Brachycladiidae Allocreadioidea 100 95.59 LC269094.1 Campula oblonga Brachycladiidae Allocreadioidea 100 95.59 KM258665.1 Pleorchis uku Acanthocolpidae Acanthocolpioidea 100 95.59 DQ248203.1 Skrjabinopsolus nudidorsalis Deropristidae Lepocreadioidea 100 95.59 MN700960.1 Pleorchis polyorchis Acanthocolpidae Acanthocolpioidea 100 95.45 DQ248202.1 Oschmarinella rochebruni Brachycladiidae Allocreadioidea 100 95.45 KM258667.1 Nasitrema sp. Brachycladiidae Allocreadioidea 100 95.45 KM258666.1 Brachycladium goliath Brachycladiidae Allocreadioidea 100 95.32 KR703279.1 Synthesium tursionis Brachycladiidae Allocreadioidea 100 95.31 FJ357163.1 Synthesium pontoporiae Brachycladiidae Allocreadioidea 100 95.31 FJ357162.1 Nasitrema globicephalae Brachycladiidae Allocreadioidea 100 95.31 AJ004968.1 Fig. 1. Phylogenetic trees based on 18S rRNA sequences using the ML method with 500 bootstrap replicates. Th e tree with the highest log likelihood (-1959,87) is shown. Th e percentage of trees in which the associated taxa clustered together is shown next to the branches. 87Molecular Characterization of the First Reported Neoplagioglyphe megastomus... ditional species revealed as the closest relatives were chosen for the phylogenetic analysis (table 3). Th e most distant species from the Acanthocolpidae were used to root obtained trees. Nucleotide sequences were aligned using the Clustal W option of MEGA-11. Trees were obtained using maximum likelihood (ML) with Tamu- ra-Nei model, neighbour joining (NJ) and minimum evolution (ME) methods as applied in MEGA-11. Clade support was assessed by bootstrap resampling with 500 replicates. Results M o l e c u l a r i d e n t i f i c a t i o n 18S rRNA sequence obtained from the metacercaria studied here is deposited in GenBank under accession numbers OM044587. BLAST analyses on this sequence showed the closest similarity with trematodes belonging to the superfamily Plagiorchioidea (Bray, 2008) (Trematoda: Digena: Plagiorchiida) (table 3). Phylogenetic analysis further confi rms that the studied here metacercariae belong to this superfamily (strongly supported by a high bootstrap value, 100 %) and is in the sister relationship with Rubenstrema exasperatum (Rudolphi, 1819) of the Omphalometridae Looss, 1899 (highly supported by a bootstrap, 90 %) (fi g. 1). Phylogenetic trees built with NJ and ME algorithms showed the same topology, but the bootstrap support for clade formed by R. exasperatum and the metacercaria sequenced in the present study was slightly higher (94 %). D i f f e r e n t i a l a n a l y s i s w i t h i n t h e O m p h a l o m e t r i d a e Th e last revision of Omphalometridae by Tkach (2008) defi ned 5 genera within the family: Omphalometra Looss, 1899, Rutshurutrema Baer, 1959, Rubenstrema Dollfus, 1949, Neoglyphe Shaldybin, 1953 and Neoplagioglyphe Tkach, 2008. Specimens of metacercariae found in the present study are morphologically similar to Neoplagioglyphe. Th ey are distinguished from Omphalometra, Rutshurutrema, Rubenstrema and Neoglyphe by the following combination of characters: i) body small, slender, elongate, narrows at posterior end; ii) oral sucker much larger than ventral; iii) testes entire, rounded or spherical, contiguous, tandem, in posterior half of body; iv) cirrus sac claviform, curved, in hindbody, extends posteriorly beyond posterior margin of ventral sucker, contains bipartite seminal vesicle, prostatic complex and ejaculatory duct, cirrus unspined; v) ovary posterolateral or lateral to ventral sucker. Whereas those features are following: i) body very elongate, ii) suckers relatively small, either equal in size or oral sucker slightly larger than ventral sucker, iii) testes deeply lobed, iv) ovary median, in mid-region of body in Omphalometra; i) body oval or elongate, ii) suckers approximately equal in size, iii) testes lobed, iv) ovary median in Rutshurutrema; i) body relatively large, very muscular and thick, ii) ventral sucker larger than oral sucker, iii) testes entire or lobed, iv) cirrus sac entirely in forebody in Rubenstrema; i) body pear-shaped or oval, extremely fl attened dorsoventrally, ii) suckers approximately similar in size or oral sucker slightly larger than ventral, iii) testes irregularly shaped, frequently lobed, strongly transversely elongate, iv) cirrus covered with very small spines in Neoglyphe. M o r p h o l o g i c a l d e s c r i p t i o n o f t h e s t u d i e d m e t c e r c a r i a e ( t a b l e 2 , f i g . 2 ) Excysted metacercariae obovoid, tapered at posterior end, small, slender, elongate, maximum width in mid-body; oral sucker subterminal and round much larger than ventral one; ventral sucker at mid-body; prepharynx very short; pharynx ovoid broadly, well developed; oesophagus indistinct; intestinal bifurcation occurs at one-fourth of body length, distant from ventral sucker; ventral sucker small, at one-third to one-half of the total length according to the condition of contraction; caeca terminate close to posterior extremity; testes mature, entire, rounded or spherical, contiguous, tandem, in posterior third of body; cirrus sac claviform, curved, reaches well into hindbody to left of ovary, contains bipartite seminal vesicle, prostatic complex and ejaculatory duct; genital pore in forebody, submedian; ovary posterolateral or lateral to ventral sucker; vitellarium comprises 88 V. Sarabeev, M. Ovcharenko, A. S. Ahmed, R. A. Sueiro, J. M. Leiro numerous follicles forming lateral fi elds confl uent posterior to testes and extends anteriorly to level of pharynx; excretory vesicle Y-shaped. Seminal receptacle and uterus were not observed. Discussion Metacercariae reported here are identifi ed as Neoplagioglyphe megastomus (Baer, 1943) based on their morphological and biological features. Th e dimensions of metacercariae and the condition of the genital glands indicate that those were mature and quite similar morphologically to the adult form. Our fi nding corroborates the results of Baer (1943), who reported metacercariae with well developed genital systems and measurements similar to or even larger than in the adults. Th e individuals revealed here were about one-third larger in the body size than those reported by Baer (1943), including both adult and larval stages (table 2). Th e larger dimensions in the body size can be explained by the fact that diff erent processing techniques were used when manipulating metacercariae. We measured alive worms, while Baer (1943) used ethanol fi xed and stained material. Th e infl uence of storage and examination methods on the size of metacercariae was investigated by Lepitzki, et al. (1994), who showed that variation between measurements of ethanol fi xed and alive worms may be substantial, reaching as high as 40 % to 50 %. Another reason that may explain metrical distinctions is the presence of two geographically isolated forms. In the body shape and measurements, specimens examined here are more similar to the form described by Matskási (1971) (e. g. fi gs 11 and 12). Th e ratios used to compare metacercariae and adults, drawn from published fi gures for the latter ones (Baer, 1943; Matskási, 1971), showed Fig. 2. Microphotographs and line drawing of live metacercariae of Neoplagioglyphe megastomus from Gammarus pulex. A. Encysted metacercaria. B. Dorsal view of ovarian complex and male terminal genitalia. C. and D. dorsal view of metacercaria removed from cyst. Abbreviations: C, cirrus; CS, cirrus sac; L, Laurer’s canal; MG, Mehlis’ gland cells; O, ovary; OO, ootype; T, anterior testis; VS, ventral sucker. 89Molecular Characterization of the First Reported Neoplagioglyphe megastomus... that those are in the range or overlapped broadly (table 2), thus further supporting our identifi cation of these worms as N. megastomus. Th e Omphalometridae is a small group of plagiorchioid digenean taxa, which combines Omphalometra, Rutshurutrema, Rubenstrema, Neoglyphe and Neoplagioglyphe (Tkach, 2008). Adults are parasites of the stomach, intestine and gallbladder of shrews, moles and desmans. Th e family is characterized by the uterus, which never entered the post-testicular region of the body and usually not beyond the anterior testis (Tkach, 2008). Although the uterus as a diagnostic feature was not observed in the present study, the family affi nity of the metacercariae was determined based on molecular information. Neoplagioglyphe megastomus was in the sister relationship with Rubenstrema comprising together the most basal clade of the monophyletic Plagiorchioidea clade. Th e BLAST search revealed 98.2 % identity for sequence of R. exasperatum with that obtained here. Th e species identity threshold for V4 and V5 regions of 18S rRNA is usually assigned at the level of 97–98 % (Aguilar et al., 2016; Choi and Park, 2020; Sarabeev et al., 2020). Th is fi nding further supports the morphological observation that specifi ed taxonomic distinctions between N. megastomus and R. exasperatum. Adults of N. megastomus were described from Eurasian water shrew, Neomys fodiens (Pennant, 1771), in streams around Neuchatel, Switzerland. Encysted metacercariae of this species were found from Gammarus pulex (L.) in the same waters in which the shrew was caught. Th e full life cycle was elucidated by Vaucher (1971), who obtained cercariae from Radix peregra (Müller, 1774) sampled in the Areuse River near Neuchatel and infected individuals of Gammarus in the experimental study. Th e literature review of geographic records of N. megastomus indicates that the distribution of this species is related to freshwater ecosystems of Europe. In addition to Neuchatel, N. megastomus was also reported from the type host in the Giessen Area, Germany (Brendow, 1970), the High Tatras, Slovakia (Prokopic, 1957); from Neomys anomalus Cabrera, 1907 in Németbány, Hungary (Matskási, 1971); from N. anomalus and Sorex araneus L. in Sopron, Hungary (Matskási, 1971; Gubányi et al., 2002). Out of 7 observed localities in the Pomeranian region of Poland N. megastomus was found in the stream close to Krępa Słupska and the Lupawa River close to Smoldzino (table 1). Th e intensity of infection reached 4 and 8 individuals per host with a prevalence 10 % and 38 % in Smoldzino and Krępa Słupska, respectively. Cystes were localized in the body cavity along the intestinal tract. Similarly, a low prevalence of infection in amphipods was reported by Baer (1943) in Neuchatel. Given the evident similarity in morphological and biological characteristics, supplemented by molecular data, it may be concluded that the metacercariae studied herein represent N. megastomus. To our knowledge, this is the fi rst report of N. megastomus in Poland and is the fi rst molecular characterization of the worm. Further molecular data is needed to elucidate relationships between geographically isolated forms that diff er in dimensions. 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