Int. J. Aquat. Biol. (2020) 8(4): 246-252 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2020 Iranian Society of Ichthyology Original Article An investigation on the helminth parasites of Caspian turtle (Mauremys caspica) with a taxonomic note on recovered Falcaustra Lane, 1915 (Nematoda: Kathlaniidae) and Spiroxys Schneider, 1866 species (Nematoda: Gnathostomatidae) Ehsan Rakhshandehroo*1, Amin Gholamhosseini2, Amin Ahmadi1, Mostafa Rakhshaninejad2, Amir Ali Heidari2 1Department of Pathobiology, School of Veterinary Medicine; Shiraz University, Shiraz, Iran. 2Department of Aquatic Animal Health and Diseases, School of Veterinary Medicine, Shiraz University, Shiraz, Iran s Article history: Received 15 April 2020 Accepted 21 August 2020 Available online 2 5 August 2020 Keywords: Caspian turtle Falcaustra Spiroxys Phylogeny Abstract: In this study, the digestive tracts of the Caspian turtles (Mauremys caspica) were investigated for the presence of helminth infections. Specimens of roundworms were recovered from the large intestine and stomach of the Caspian turtles. The morphologic measures revealed the infection with nematodes of the genus Falcaustra and Spiroxys. However, some differences were found in the collected Falcaustra specimens compared to the previous descriptions. In parallel, samples were analyzed by sequencing of the ribosomal gene targets. The phylogenetic analysis showed that the Falcaustra species as members of superfamily Cosmocercoidea were grouped with some members of Ascaridoidea and Spiruroidea. Despite the significant morphologic differences, Spiroxys species formed a sister group with ascaroid and cosmocercoid roundworms. Therefore, our molecular findings revealed that the taxonomic position of both nematodes need be revised. Introduction Nematodes of the genus Falcaustra Lane, 1915 and Spiroxys Schneider, 1866 have been reported from different species of turtles around the world. Bursey et al. (2000) listed a total of 68 Falcaustra species in reptilian hosts from which 29 species infect turtles. In recent two decades, a number of new species of Falcaustra were introduced from the digestive tract of turtles (Bursey and Kinsella, 2003; Bursey and Rivera, 2009; Bursey and Brooks, 2011) and other reptiles (Bursey et al., 2007; Bursey et al., 2009). Spirurid nematodes of the genus Spiroxys are also parasitic in the alimentary canal (mainly stomach) of this type of hosts (Baker, 1987) with more than 17 recorded species (Roca and García, 2008). Most of the taxonomic knowledge on both above- mentioned genera are based on morphological features. This has been led to describing many new taxa that are often difficult to identify (Baker et al., 1986). In addition, the taxonomy of the genus Falcaustra is somewhat confused because of several provided descriptions of some species and the fact that *Correspondence: Ehsan Rakhshandehroo E-mail: rakhshandehroo@shirazu.ac.ir the information on some species is inadequate. Although molecular-based methods have developed for the identification and taxonomy of helminths, data for molecular diagnosis of Falcaustra and Spiroxys is rare (Li et al., 2014; Rajabloo et al., 2017). Therefore, the integration of genetic markers with morphology could be important for accurate identification. In the present study, the helminths recovered from the stomach and large intestine of the Caspian turtle (Mauremys caspica) (Testudines: Geoemydidae) were examined by conducting a morphological study and molecular analyses of their targeted regions in ribosomal RNA gene. Materials and Methods The parasite specimens and diagnosis: A total of 5 infected turtles of M. caspica were collected in the Sepidan (29°54'27"N 52°32'48"E) and Beyza (29°58'19"N 52°24'4"E) regions, Fars Province, southern Iran. Turtles were subjected to standard euthanasia and post mortem examination using protocols described by Mader and Garner (2006) and 247 Int. J. Aquat. Biol. (2020) 8(4): 246-252 Underwood and Reymond (2020). The alimentary canal including the esophagus, stomach, small and large intestines of turtles were examined separately for the presence of helminthes. Some of the recovered specimens were preserved and fixed in 70% ethanol, cleared in lactophenol, mounted and examined for morphological examinations according to Bursey et al. (2009) and some were fixed into 96% ethanol for molecular study. Molecular procedures: Genomic DNA was extracted from the adult nematodes using a commercial Kit (MBST, Iran) according to the manufacturer’s protocol. Our investigation showed infection of the examined turtle specimens with the species of Falcaustra and Spiroxys in the large intestine and stomach, respectively. The search for the recorded Falcaustra in the GenBank® revealed few available items mainly ribosomal and also mitochondrial (Cytochrome Oxidase) genes. On the basis of sequences for the partial 18s rDNA, a pair of primers: F (5'-AGAAACGGCTACCACATC-3') and R (5'- TTACGGTCAGAACTAGGG-3') were designed and used for recovered Falcaustra sp.. The PCR condition was 94°C for 4 min (initial denaturation), followed by 35 cycles of 94°C for 1 min (denaturation), 54.6°C for 1 min and 72°C for 1 min (extension) and a final elongation at 72°C for 2 min. For molecular diagnosis of the stomach worms i.e. Spiroxys sp., the partial 18s rDNA and internal transcribed spacer (ITS) regions were considered and amplified using primers: F (5'-AGAGGT GAAA TT CGTGGACC-3') and R (5'-ATATGCTTAAGTTCA GCGGGT-3') (Hasegawa et al., 2009). PCR condition was 94°C for 4 min (initial denaturation), followed by 35 cycles of 94°C for 1 min, 55.7°C for 1 min and 72°C for 1 min (extension) and a final elongation at 72°C for 25 min. The PCR reactions were adjusted in a final volume of 25 μl, including 12.5 μl of PCR premix (Ampliqon, Denmark, Cat. No. A180301) (containing Tris-HCl pH: 8.5, (NH4)2S04, 3 mM MgCl2, 0.2% Tween 20, 0.4 mM of each dNTP, 0.2 units/µl Taq DNA polymerase and Inert red dye and stabilizer), 10 pmol of each primer, 6.5 μl H2O and 4 μl of DNA extracted as template. The presence of the expected amplicons and their size were assessed by electrophoresis on each reaction product in 1.2% (w/v) Tris-acetate/EDTA agarose gel and visualized under ultraviolet illumination. Products from each assay were sequenced (ABI 3730 DNA analyzer; Bioneer, Korea). Because the specimens were found for the first time in the region, no positive control was available to conduct in molecular assay. To determine the phylogenic positions of recovered parasites, the sequences were compared with previously reported other nematodes in the GenBank. Creating multiple-sequence alignment was performed using the ClustalW program embedded in the MEGA 6.0 software. Data was also used for construction of the phylogenetic trees using maximum likelihood method (Tamura et al., 2007). Results Morphologic measurements: The large intestines of all turtles were highly infected with round worms (Falcaustra sp.); however only three nematodes (one intact female and two broken) (Spiroxys sp.) were recovered from a gastric section. According to the morphologic characters, nematodes from the large intestine were belonged to the Family Kathlaniidae Lane, 1914, the genus Falcaustra (Bursey et al., 2009). Those worms have a cylindrical body tapered gently towards the tail end. At the anterior margin, the mouth opening bounded by lips, each with conspicuous pairs of large fork-like papillae (Fig. 1b). The mouth opens into the pharynx with relatively thick walls. The esophagus has subspherical isthmus and a spherical bulb at the end (Figs. 1a, c). In males (n=18), length was 15.6±1.4 (13.2-17.2 mm) and the width (at level of esophageal-intestinal junction) was 420.7±33 (372.2-459.9 µm). The esophagus consisting of vestibule (pharynx) 74.3±6.2 (65-80 µm); corpus 1939.7±236 (1608.5-2256.3 µm); isthmus 154.1±12.8 (133.2-165 µm) and a valved bulb 222.9±27.4 (173.4-244.3 µm) in length. Nerve ring was 369.4±6.5 (363.9-376.6 µm) from the anterior end. One pseudosucker was supported by 18-20 pairs of the muscle bands terminating on the border of the structure was seen in all males (Fig. 1d). Pairs of the 248 Rakhshandehroo et al. / Helminth parasites of Caspian turtle obliquely arranged muscle bands were observed beginning near posterior lip of the pseudosucker and terminating anterior to the cloaca. Five pairs of the caudal papillae, including one pair precloacal, one pair adcloacal and three pairs postcloacal (3-1+1) were observed. Spicules were similar in shape, curved, alate (which extended almost to the blunt end of the spicule), distal end pointed, 1.68±0.16 (1.48-1.9 mm) in length. Gubernaculum 178.9±17.9 (154.1-202.7 µm) in length, blunt distal tip. Tail (from the anus to the posterior end) was conical, terminated with a relatively sharp end, 574.5±68.7 (488.9-678.2 µm) in length (Fig. 1e). Females’ (n=15) length was 15.1±1.3 (13.2-17.6 mm) with width (at level of esophageal-intestinal junction) of 374.1±38.1 (319.9-455.1 µm). The esophagus consisting of vestibule (pharynx) 86.4±8.9 (70-100 µm); corpus 1689.5±163.4 (1510.1-2074.7 µm); isthmus 159.5±22.9 (138.2-212.3 µm) and the valved bulb 216.8±32.2 (174.5-288.9 µm) in length. Nerve ring was 416.8±67.8 (367.5-478.1 µm) from the anterior end. Vagina directed anterodorsally giving rise to two divergent amphidelphic uteri. Vulva, slightly prominent slit, 5.3±0.3 (5-5.4 mm) from the posterior end; eggs were oval to elliptical, thick shelled, unembryonated and 114.8±9.8 × 69.7±6 in size. Tail was conical, 1098.9±106.2 (976.5-1164.9 µm) (Figs. 2a, b). Major morphologic measurements of Falcaustra species reported having one pseudosucker structure are listed in Table 1. According to described features, Figure 1. Falcaustra sp. (the Iranian isolates); (a) The anterior end (dorsal view); (b) The head region, showing large forked papillae on the head (arrows); (Ph) pharynx (vestibule) and proximal part of the esophagus (Eso); (C) Esophagus (distal part). isthmus (is) and bulbus (b) parts; (d) Male, the pseudosucker structure showing radiating muscles (arrow); (e) Caudal end of male. Oblique muscles (om); Caudal papillae (cp) spicules and gubernaculum (gub). Scale bar~300 µm. Figure 2. Falcaustra sp., female. (a) Distal part of reproductive system; (b) female, conical tail (arrow points to the anus); (c) Spiroxys sp., anterior part include the esophagus, muscular (m) and glandular (g) parts; (d) Trilobed pseudolabia forming a tooth on median part (arrow); (e) The female reproductive system; (f) Caudal end of female, arrow points to the anus. Scale bar~300 µm. 249 Int. J. Aquat. Biol. (2020) 8(4): 246-252 our specimens were identified as F. ararath Massino, 1924, described from a species of fresh water turtle, Emys orbicularis, in Armenia however, differences are seen in the papillae pattern and the average spicule size in males. In this study, only one intact female nematode of Table 1. Main (selected) characterizations of some species of Falcaustra which are relatively closed to the specimens recovered in the present study. Species Host Body length (mm) Spicule length (mm) Pseudo sucker Papillae pattern Country Falcaustra ararath (current study) Caspian turtle (Mauremys caspica) 15.3 1.68 (1.48-1.9) 1 3-1 +1 Iran F. ararath (Massino, 1924) European pond turtle (Emys orbicularis) 15.0 1.54 1 8–6–10 +1 Armenia F. kaveri (Karve and Naik, 1951) cyprinid fish (Barbus carnaticus) 13.7–15.5 2.00–2.23 1 6–0–16 +1 India F. armenica (Massino, 1924) European pond turtle (Emys orbicularis) 7.2–8.9 1.10–1.30 1 6–2–10 Armenia F. dubia (Yuen, 1963) fanged river frog (Limnonectes macrodon) 13.5–14.0 1.54–1.69 1 6–2–12 +1 Malaysia F. kempi (Baylis & Daubney, 1922) Elongated tortoise (Indotestudo elongate) 10.9–12.8 2.90 1 10–0–8 +1 India F. simpsoni (Johnston and Mawson, 1944) Bell frog (Litoria aurea) 13.0–13.5 2.00 1 8–0–12 +1 Australia F. chelydrae (Harwood, 1932) Common snapping turtle (Chelydra serpentina) 10.0–12.5 3.4–3.9 1 6–0–14 USA Figure 3. Phylogenetic position of the Falcaustra (a) and Spiroxys (b) isolates from Caspian turtles among nematode species in different hosts inferred from partial ribosomal gene targets using the maximum-likelihood method. Bootstrap values (2000 psuedoreplicates) represented at the nodes. The horizontal distance is proportional to evolutionary change as indicated (scale bar). 250 Rakhshandehroo et al. / Helminth parasites of Caspian turtle the genus Spiroxys was found in the stomach content and investigated. The morphology was indicative of the genus Spiroxys Schneider, 1866 (Figs. 2c-f). Its body length is 27.4 mm, the maximum width 676.5 µm, the oesophagus (2543.2 µm), thickened from the first third to the posterior and divided into anterior muscular, and a long posterior glandular portion (Fig. 2c). The cephalic end surrounded by the trilobed pseudolabia projectinh anteriorly in each median lobe to form a tooth (Fig. 2d). Thin-shelled sub-elliptical eggs were seen with a mean length of 56.96±6.4 × 41.04±6.6 µm (Fig. 2e). Tail was short, stout and conical, 333.18 µm in size. According to these features, our specimen was identified as Spiroxys ankarafantsika, described by Roca and García (2008) from Madagascan pleurodiran turtles (Pelusios castanoides and Pelomedusa subrufa). Molecular analysis: The sequences of ribosomal gene targets were detected in samples and successfully amplified. For Falcaustra samples, DNA fragments of about 600bp was obtained for partial 18s rRNA gene (recorded in the GenBanks as MT160412). The comparison between our data and the three available sequences for Falcaustra in the GenBank revealed about 98.4 percent identity. Our samples had also a high identity with two other nematodes, Paraquimperia Africana (the intestinal parasite of a eel species) (98.4%) and Ichtyobronema hamulatum from burbot, Lota lota (97.9%). The phylogenetic analysis confirmed the close relationship between our samples and F. araxiana and F. catesbeianae separated from turtle and frog hosts, respectively (Fig. 3a). It was unexpected that the Falcaustra species as members of superfamily Cosmocercoidea were grouped with members of superfamilies Ascaridoidea (Paraquimperia) and Spiruroidea (Ichtyobronema); however, the other cosmocercoid nematodes were located in a separate clade. Furthermore, our specimens formed a sister group with some members of Seuratoidea (Cucullanus, Dichelyne and Truttaedacnitis). The targeted ribosomal region was amplified in Spiroxys genetic material leading to fragments of about 1140 bp. Our specimens had nearly complete identity with S. japonica (AB818381 and AB818382) and S. hanzaki (AB818383) found in frog (Lithobates catesbeianus) and salamander (Andrias japonicus) hosts, respectively. In addition, a great identity was observed between our specimens, as members of Gnathostomidea, with Anguillicola crassus from superfamily Dracunculoidea. According to the phylogenetic tree, both nematodes are grouped with each other (Fig. 3b). Based on the ribosomal region, Spiroxys sp. formed a sister group with ascaroid and cosmocercoid nematodes, whereas there are significant differences between those taxa according to the morphologic characteristics. Discussions Different species of nematodes have been described occurring in the digestive tract of turtles (Bursey et al., 2009; Bursey and Brooks, 2011). Of the prevalent roundworms, the infection with the species of Falcaustra and Spiroxys were found in Caspian turtle. Previously, F. armenica from Caspian turtles (Youssefi et al., 2015) and F. araxiana from European pond turtles (Emys orbicularis) (Rajabloo et al., 2017) were reported in north and southwest regions of Iran, respectively. Unexpectedly, despite the similarity of the host, our samples had different morphology compared to those species. This indicates the diversity of the parasite in different localities. Having similar turtle hosts, F. ararath, F. araxiana and F. armenica were reported from Emys orbicularis in Armenia (the northwest neighbor of Iran), F. donanaensis from Mauremys leprosa in Spain (Hidalgo-Vila et al., 2006) and F. manouriacola from impressed tortoise (Manouria impressa) (Bursey et al., 2009). All the species had differences in main morphologic features, including the body size, spicule length and the number of pseudosuckers. In this study, the structure of the esophagus, head and presence of one pseudosucker agreed with those previously described F. ararath Massino, 1924. In contrast, the caudal papillae pattern was not exactly unique to the previous description. In addition, regarding the average spicule length, our specimens had relatively higher measurements (1.68 comparing 251 Int. J. Aquat. Biol. (2020) 8(4): 246-252 to 1.54 µm). Nonetheless, these differences were not significant enough to assign our isolates into a new taxon. Although numbers of species have explained in pervious literature for the genus Falcaustra, many of reports have re-described them or introduced new species during the past decades. In turtles, in addition to 29 species listed by Bursey et al. (2000), several new species have also assigned as F. manouriacola from M. impressa (Bursey and Rivera, 2009), F. kutcheri from Geoemyda yuwonoi (Bursey et al., 2000), F. greineri from Orlitia borneensis (Bursey and Kinsella 2003) and F. donanaensis from M. leprosa (Hidalgo-Vila et al., 2006). Regarding the controversial and/or overlapping morphological measurements in the past, the taxonomic investigation of those species is difficult. This was also the case for the Spiroxys samples. Although we did not recover male worms, the only recovered female had characteristics close to S. ankarafantsika separated from Madagascar freshwater turtles (Roca and García, 2008). Many of the Spiroxys species have found in fresh water turtles within the suborder Cryptodira (Berry, 1985). The Caspian turtles belongs to Cryptodirans, however, S. ankarafantsika separated from pleurodiran turtles (belong to suborder Pleurodira) (Roca and García, 2008). According to reports on the presence of Spiroxys spp. in pleurodiran turtles in Australian (Berry, 1985), Madagascan (Roca et al., 2007) and Ethiopia (Berry, 1985), it was hypothesized that this nematode has been acquired from non-marine cryptodirans (Roca et al., 2007). Terefore, if we consider our samples as a type of S. ankarafantsika, it can be concluded that the transfer of Spiroxys sp. between two suborders of fresh water turtles have previously occurred. Despite the presence of accurate morphologic characterization, molecular data on Falcaustra and Spiroxys is rare. In the GenBank, few records exist for F. araxiana and F. catesbeianae and for S. japonica and S. hanzaki. Nevertheless, on the basis of partial ribosomal gene, the cosmocercoid nematodes formed a separate clade; however, the Falcaustra species of turtles, as representatives of cosmocercoidea, were grouped with Paraquimperia (of Ascaridoidea) and Ichtyobronema (from Spiruroidea), both separated from kinds of fish. These groups possess significantly different morphology at least on their head region. In addition, a great identity was observed between the Spiroxys, as member of Gnathostomidea, with Anguillicolaoïdes crassus from Dracunculoidea and with ascaroid and cosmocercoid nematodes, both with a distinct structure. We found also the close relationship between F. araxiana and Paraquimperia africana showing the ambiguity on the present classification and phylogenetic relationship (Rajabloo et al., 2017). This controversial maybe because partial sequences have been considered and the number of available records is too limited to have an accurate molecular comparison. In this study, the sequence data for the Spiroxys specimen was so close to S. japonica, but the morphologic characters were not agreed with this species well. On the other side, it was stated that the 18S rDNA and ITS regions revealed significant nucleotide differences comparing the two prevalent species, S. japonica and S. hanzaki (Li et al., 2014). Hence, it seems that the selected regions could be used for the identification at the level of species instead of the genus or higher taxa. As a conclusion, these results criticize the morphologic data. 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