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JOURNAL of NEMATOLOGY Morphological and Molecular JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2021-048 e2021-48 | Vol. 53 Morphological and molecular characterisation of Tripylina gorganensis from the Slovak Republic as a contribution to the redescription of the species Marek Renčo1,*, Katarzyna Rybarczyk-Mydłowska2, Łukasz Flis2, Magdalena Kubicz2 Abstract 2 and Grażyna Winiszewska Specimens of Tripylina gorganensis, collected from a natural beech 1Institute of Parasitology, Slovak forest in Slovak Republic, are described and illustrated. These Academy of Sciences, Hlinkova 3, nematodes were initially identified as an undescribed species, Košice, Slovak Republic. morphologically similar to Tripylina gorganensis described from Iran. An important feature distinguishing both species was the presence 2Museum and Institute of Zoology of post-vulval uterine sac (PUS) in specimens from Slovak Republic, PAS, Wilcza 64, 00-679 Warsaw, which, according to the original description (Asghari et al., 2012), was Poland. absent in Tripylina gorganensis. However, a careful re-examination of *E-mail: [email protected] type specimens performed in this study revealed that T. gorganensis also has the post-vulval uterine sac. Consequently, the findings of All authors contributed equally to the morphological and molecular studies performed on the Slovak this study. population and observations on the type material contribute to the This paper was edited by redescription of T. gorganensis. Thomas Powers. Received for publication Keywords October 14, 2020. Redescription, Fagus sylvatica, Molecular, Morphology, Morphometrics, Phylogeny, Ribosomal DNA, Taxonomy, Tripylina gorganensis. The genus Tripylina Brzeski, 1963 is represented de Man, 1880 rather than Tripyla Bastian, 1865, by free-living, predacious species that inhabit soil, Tripylella Brzeski & Winiszewska-Ślipińska, 1993 or litter, moss, and other semi-wet biotopes (Andrássy, Tobrilus Andrássy, 1959 (Tripylidae, 62 Triplonchida) 2007) and are often found in close proximity to supporting Andrássy’s (2007) view that Tripylina various tree species (Asghari et al., 2012; Xu et al., could be subsumed under Enoplida, rather than 2013; Zhao, 2009). This genus currently includes the Tripylidae de Man, 1876 associated with the 22 species described from different regions of the Triplonchida (Cid del Prado-Vera et al., 2012, 2016; world (Table 1). Zhao, 2009, 2011). Starting with the work by Zhao (2009), half of The present work focuses on morphological and the described species have been characterized molecular characterisation of the Tripylina gorganensis molecularly by partial sequences of either one or found during an ecological study, on the impact of two of the ribosomal DNA genes (18 S and/or 28 S natural disturbance on soil nematode communities’ rDNA). The rDNA-based phylogenetic analyses structures in a natural beech forest (Fagus sylvatica revealed the well-supported monophyletic status L.) in the Slovak Republic. Re-examination of the of Tripylina (Asghari et al., 2012; Cid del Prado-Vera paratypes of T. gorganensis has pointed out the et al., 2012, 2016; Xu et al., 2013; Zhao, 2009). The necessity to redescribe this species. T. gorganensis Tripylina clade was shown to be more closely related happens to be another European species follo- to the Enoplida representatives like Pseudocella wing the reports of T. arenicola, T. longa, and Filipjev, 1927, Deontostoma Filipjev, 1916 or Alaimus T. macroseta. © 2021 Authors. This is an Open Access article licensed under the Creative 1 Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ Morphological and molecular characterisation of Tripylina gorganensis from the Slovak Republic: Renco et al. Table 1. Alphabetical list of Tripylina species and country name from which they were originally described. Species Country T. arenicola (de Man, 1880) Brzeski, 1963 The Netherlands T. bravoae Cid del Prado Vera et al., 2012 Mexico T. gorganensis Asghari et al., 2012 Iran T. iandrassyi Cid del Prado-Vera et al., 2016 Mexico T. ixayocensis Cid del Prado Vera et al., 2012 Mexico T. kaikoura Zhao, 2009 New Zealand T. longa Brzeski & Winiszewska-S´ lipin´ska, 1993 Italy T. macroseta (Vinciguerra & La Fauci, 1978) Tsalolikhin, 1983 Italy T. manurewa Zhao, 2009 New Zealand T. montecilloensis Cid del Prado Vera et al., 2012 Mexico T. puxianensis Xu et al., 2013 China T. rorkabanarum Cid del Prado-Vera et al., 2016 Mexico T. sheri Brzeski, 1963 USA (California) T. stramenti (Yeates, 1972) Tsalolikhin, 1983 New Zealand T. tamaki Zhao, 2009 New Zealand T. tearoha Zhao, 2009 New Zealand T. tlamincasensis Cid del Prado Vera et al., 2012 Mexico T. ursulae (Argo & Heyns, 1973) Tsalolikhin, 1983 South Africa T. valiathani Tahseen & Nusrat, 2010 India T. yeatesi Zhao, 2009 New Zealand T. ymyensis Tahseen & Nusrat, 2010 China T. zhejiangensis Pham et al., 2013 China Material and methods processed by the Cobb sieving and decanting method (Cobb, 1918) followed by the modified Baermann Soil sampling, nematode extraction, technique (van Benzooijen, 2006). Subsequently, and processing nematodes were extracted from the aqueous soil suspensions using a set of two cotton-propylene Soil filters. Suspensions containing nematodes were collected after 24 h. The nematode suspensions were In total, ten representative soil samples composed subsequently examined under a stereomicroscope from five subsamples were collected from the five (40 × and 60 × magnification). After the excess water permanent research plots (10 × 10 m) established in the was removed, five Tripylina individuals was set aside undisturbed natural beech forest in Opátka (48°47’N, for molecular analyses and therefore placed in an 21°04’E, at 776 m a.s.l.), a small village in Košice district, Eppendorf tube containing water and subsequently eastern Slovakia. Samples were collected twice, five in sent to Poland. The remaining nematodes were used May 2016 and five in September 2018, and examined to morphological and morphometric study, fixed in two laboratories, first in Slovakia and later in Poland. with a hot 99:1 solution of 4% formaldehyde and Each sample (collected in 2016) was homogenized pure glycerin (Seinhorst, 1962) and then processed by gentle hand mixing. A total of 100 g of soil was to anhydrous glycerin (Seinhorst, 1959). Permanent then soaked in 1 l of tap water for 30–60 min and slides were made and Tripylina nematode individuals 2 JOURNAL OF NEMATOLOGY were studied using a light microscope Eclipse 90i 94°C for 3 min, followed by 35–40 cycles at 94°C Nikon, Japan at 100, 200, 400, 600, and 1000 × for 30 s, 54°C (58°C in case of D2A-D3B primer magnification. combination) for 30 s and 72°C for 70 s with a final The soil samples collected in 2018 were extracted incubation for 5–7 min at 72°C. Amplicons were in Poland by the decantation and sieving method visualised under UV illumination after Midori Green followed by the centrifugal flotation method (van (Nippon Genetics Europe, Duren, Germany) or Simply Benzooijen, 2006). Extracted nematodes were heat- Safe (EURx, Gdańsk, Poland) gel staining and gel killed using tap water at 60°C. Some of the selected electrophoresis. Excess dNTPs and unincorporated individuals (as well as a few individuals from 2016 primers were removed from the PCR product using that arrived alive in water from Slovakia) were fixed the Clean-Up Purification Kit (A&A Biotechnology, in TAF and subsequently submitted for morphologi- Gdynia, Poland) or alternatively the enzymatic mixture cal analysis. Photographs and measurements were of Fast Polar-BAP – Thermosensitive Bacterial Alka- taken from 16 females and 14 males with a Leica line Phosphatase (EURx, Gdańsk, Poland) and Exo- DFC 500 camera on a Leica DM 5000B microscope. nuclease I – (EURx, Gdańsk, Poland) was used. The remaining nematodes were fixed in DESS and Both the 18 S and 28 S rDNA fragments were temporary slides were prepared. With photographic successfully amplified and sequenced from all 10 documentation collected, the examined specimens processed nematode individuals. There were no were washed with PBS and subsequently with sterile sequence variations, neither in the 18 S rDNA, nor water and then submitted for molecular studies. within the 28 S rDNA region. The obtained sequence fragments of 18 S rDNA and 28 S rDNA were de- Nematode lysis, amplification, and posited in GenBank under the following accession numbers: MK722523 and MK733311. sequencing of 18 S and 28 rDNA fragments Phylogenetic analysis Ten selected individual nematodes (five collected in 2016 and five in 2018) were transferred to separate The newly obtained sequences of Tripylina (18 S and 0.2 ml polymerase chain reaction (PCR) tubes 28 S rDNA fragments) were analyzed with publicly containing 25 μ l sterile water. An equal volume of the available (GenBank) rDNA sequences, using the BioEdit lysis buffer, as described in Holterman et al. (2006), program (v. 7.2.5; Hall, 1999). The 18 S rDNA dataset was added. The lysis occurred in a Thermal cycler included the Enoplida sequences from representatives (Veriti 96-Well Thermal Cycler, Applied Biosystems, of the genera: Tripylina, Trischistoma, Trefusia de Foster City, CA, USA) at 65°C for 3 h followed by Man, 1893, Bathylaimus Cobb, 1894, Deontostoma, a 5 min incubation at 100°C. The obtained single Phanoderma Bastian, 1865, Pseudocella, Thora- nematode lysate (crude DNA extract) was either used costoma Marion, 1870, Rhabdolaimus de Man, immediately as a DNA template for a PCR reaction or 1880, Syringolaimus de Man, 1888, Campydora stored at
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