OSWALDOCRUZIA FILIFORMIS SENSU LATO (NEMATODA: MOLINEIDAE) FROM AMPHIBIANS AND REPTILES IN EUROPEAN RUSSIA: MORPHOLOGICAL AND MOLECULAR DATA

Nematodes of the genus Oswaldocruzia parasitise the small intestine of amphibians and reptiles. Their biodiversity remains unknown. We studied Oswaldocruzia nematodes from nine species of amphibians and reptiles (Pelophylax ridibundus, Rana arvalis, Rana temporaria, Bufo bufo, Lacerta agilis, Zootoca vivipara, Anguis fragilis, Natrix natrix, Vipera berus) at six localities in European Russia in 2018–2019. To identify their nematode species, we analysed the morphological characters traditionally used in the taxonomy of nematodes of this genus together with new molecular phylogenetic data. The results of partial sequencing and molecular phylogenetic analysis of CoxI mtDNA gene showed that all Oswaldocruzia specimens in this study belonged to the same species. We observed a broad morphological variability of nematodes both from different host species and from the same host individual. Morphological variation in nematodes from various host species could be host-induced, while that in nematodes from the same host individuals could be due to phenotypic plasticity of the species. Molecular data indicate that only one species of the genus Oswaldocruzia, O. filiformis s.l., which has a broad morphological variability, parasitises amphibians and reptiles in European Russia. The results of our study highlight the necessity of studying the diversity of morphologically similar Oswaldocruzia spp. from the Western Palearctic by molecular genetic methods.

Oswaldocruzia filiformis Goeze, 1782 was the first species of the genus described in the Palearctic, from amphibians of the genera Bufo and Rana. The life cycle of O. filiformis is direct. Its invasive larvae occur on soil or on plants (Hendrikx & van Moppes, 1983;Tarasovskaya, 2009;Svitin, 2016). Amphibians and lizards are infected with these nematodes when they occasionally ingest their larva along with the food. Findings of O. filiformis in snakes should be considered as cases of postcyclic parasitism (Bertman & Okulewicz, 1987;Kirillov, 2000Kirillov, , 2010Novokhatskaya, 2008;Svitin & Gorobchishin, 2015;Svitin, 2016). Snakes be-come infected after eating amphibians, which are the main hosts of Oswaldocruzia spp.
The purpose of this study was to combine morphological and molecular phylogenetic data to solve the question of the species affiliation of the Oswaldocruzia nematodes parasitising different amphibian and reptilian hosts in several geographically distant localities of the European Russia.

Material and Methods
Oswaldocruzia nematodes were collected from the small intestine of amphibians and reptiles in six localities in the European part of Russia in 2018 and 2019 (Fig. 1).
The hosts of the nematodes collected in this study belonged to nine species of eight genera of amphibians and reptiles, namely: Pelophylax ridibundus (Pallas, 1771)

Statement of the welfare of animals
Our study was conducted in compliance with ethical standards of humane handling of animals. The animals were collected and processed according to the recommended practices described in Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. The amphibians and reptiles collected in this study belong to widespread and abundant species. Many of them were dead by the time of collection (dead from natural causes, road-killed or killed by rural residents or their pets). Some specimens were provided by the researchers from the IEVB of the RAS, Mordovia State Nature Reserve, National Park «Smolny» and National Park «Samarskaya Luka».
Twenty nematode specimens were recovered from amphibians or reptiles and preserved in 96% ethanol for further molecular phylogenetic analysis. For the morphological examination the nema-todes were killed by heating in water and cleared in lactic acid. In total, we studied and measured 189 specimens of Oswaldocruzia filiformis, of which 99 were females and 90 were males. The data on the geographic origin and the final hosts of the studied nematodes are provided in Fig. 1 and Table 1.
Drawings of nematodes were made using an MBI-9 light microscope with the Levenhuk M500 BASE Digital Camera and drawing tube RA-7. All the measurements are given in mm. Transverse and apical sections of nematodes were made manually with a razor blade. The synlophe was studied according to Durette-Desset (1985). The nomenclature of the caudal bursa followed Durette-Desset & Chabaud (1981). The number of dissected vertebrates, prevalence (P, %), mean abundance of helminths (A, specimens) and range (R) are given to estimate the infection of amphibians and reptiles with the parasites. If the geographical co-ordinates of the study sites are not indicated in the papers, we characterised them by their mean geographi-mean geographical coordinates obtained using the Geocode Finder (https://www.mapdevelopers.com/geocode_tool. php). All Oswaldocruzia filiformis vouchers were deposited in the parasitological collection of Saint Petersburg State University (Russia). Isolate numbers are given in Table 1.

DNA extraction, amplification and sequencing, phylogenetic analysis
The JB3/JB4.5 primer pair (Bowles et al., 1992) was successfully used for barcoding and partial phylogenetic reconstruction in species of Ancylostoma Dubini, 1843 (Hu et al., 2016) and in species identification and phylogenetic reconstruction of nematode parasites of Lissotriton vulgaris Linnaeus, 1758 (Sinsch et al., 2019). In order to obtain par-In order to obtain partial CoxI mtDNA sequences, specimens of ethanolfixed Oswaldocruzia filiformis were dried at 37°C in dry block heater for 3 h. Then the specimens were moved to clear 500 µl tubes with a mixture of 49 µl 0.1% Chelex-100 and 1 µl Proteinase K (concentration 10 mg/mL) and incubated for 3.5 h at 55°C and 25 min. at 95°C to stop proteinase activity. After that, the water solution of the total DNA was moved to sterile 500 µl tube and frozen at -80°C.
The newly obtained sequences were aligned against the full mitochondrial genome of Trichostrongylus vitrinus (GenBank no. NC_013807.1) using Muscle algorithm as implemented at CIPRES Portal (Miller et al., 2010). According to the nucleotide numbering of T. vitrinus mitochondrial genome, the newly obtained sequences were located between positions 720-1162. Other Oswaldocruzia sequences available in GenBank were located between nucleotides 49-741 (the numbering is given according to the same mitogenome no. NC_013807.1). Based on the results of this preliminary alignment, we excluded the sequences of the genus Oswaldocruzia obtained by other authors from further phylogenetic analysis.
The sequences were mounted in general alignment with other nematode species ( Table 1). The sequences were automatically aligned using Muscle algorithm (Edgar, 2004) as implemented in SeaView 4.0 (Gouy et al., 2010); the alignment was then trimmed manually. The phylogenetic analysis was performed using the maximum likelihood method at Cipres portal (Miller et al., 2010) with GTR + G + I model and a non-parametric bootstrap with 1000 pseudoreplicates. Bayesian analysis was performed with the help of MrBayes on XSEDE 3.2.7a, the GTR model with gamma correction for intersite rate variation (eight categories) and the covarion model were used. Trees were run as two separate chains (default heating parameters) for 15 000 000 generations, by which time they had ceased converging (the final average standard deviation of the split frequencies was less than 0.01). The quality of the chains was estimated using built-in MrBayes tools and, additionally, using Tracer 1.6 package (Rambaut et al., 2018); based on the estimates by Tracer, the first 6000 generations were discarded for burn-in.

Results
The nematode Oswaldocruzia filiformis s.l. was found in the samples taken from all nine amphibian and reptilian species examined in our study. The infection indices are shown in Table 2.
Among amphibians, the highest rates of infection with nematodes were registered in Rana arvalis. The infection of Bufo bufo and Rana temporaria was comparatively lower. The highest rates of nematode infection in reptiles were recorded in Lacerta agilis, while the lowest were recorded in snakes ( Table 2).

Description of nematodes
General morphology Body is thin, elongated, with maximum width at mid-length. Cephalic vesicle presents on cuticle of anterior end. Cephalic vesicle is variable in shape: whole (undivided) or consisting of two parts, a wider anterior part and a narrow posterior part, the latter smooth or with transverse folds (Fig. 2C, Fig. 3A). Vesicle shape is variable even in nematodes from one host individual. Cuticle forms uninterrupted longitudinal crests beginning behind cephalic vesicle and running along entire body. Crests are invisible on ventral side of body on transverse sections of anterior part of oesophagus region in some nematode individuals.  Anterior end is rounded. Oral opening is triangular, surrounded by four large cephalic papillae and six not always distinguishable externo-labial papillae (Fig. 2B, Fig. 3C). Oesophagus is thin, club-shaped, cylindrical in anterior part and widening posteriorly. Posterior end of oesophagus is rounded, forming posterior bulb ( Fig. 2A). Position of excretory pore varies within posterior third of oesophagus. Nerve ring surrounding oesophagus in the middle part is somewhat closer to its anterior third ( Fig. 2A).

Male
Body ends with wide caudal bursa. Caudal bursa is symmetrical, three-lobed, belonging to type II according to Durette-Desset & Chabaud (1981). Rays 2 and 3 are joined along their entire length: ray 4 is joined to ray 5 in its proximal part; rays 5 and 6 are joined along their entire length; rays 6 and 8 are joined at mid-length; rays 9 and 10 form a wide dorsal ray. Rays 9 are always with S-shaped bend. Ray 10 is always with extra branch of variable size (Fig. 2H, Fig. 3E, Fig. 5D). Only one male from Bufo bufo was with no extra branches on rays 10 (Fig. 2F), and one male from Lacerta agilis was with third extra branch on inner side of one ray 10. Genital cone is well developed, with two papillae (Fig. 2D). Gubernaculum is absent. Spicules are ap-pproximately equal, long, surrounded by thin mem-equal, long, surrounded by thin membrane, with three branches: blade is divided into two branches at the end; distal parts of each branch split into two thin branches, which are not always clearly visible; fork is divided into two branches approxi-; fork is divided into two branches approximately at mid-length. Shoe is with thin branch at mid-length and bent at distal end (Fig. 2E, Fig. 3D,  Fig. 5C). Shoe shape slightly varies in different individuals. Morphometric measurements are presented in Table 3 and Table 5.       Zootoca vivipara 2 (10 specimens)

Female
Body is larger than in males, always tapering to elongated tail with needle-shaped tip (Fig. 2F, Fig.  3B). Vulva is wide, opening with a transverse slot postequatorially (behind mid-length of female body). Muscular vagina passes into paired ovejector with powerful sphincters (Fig. 2G). Anterior ovary forms numerous loops and bends in anterior part of body, reaching oesophagus level and returning back. Posterior ovary extends to the end of the body, bending strongly and extending forward with slight twisting, going beyond vulva level. Uterus of mature females is filled with eggs. All eggs were found in uterus, ovejector and vulva at morula stage. Morphometric measurements are given in Table 4 and Table 6.

Molecular phylogenetic analysis
All Oswaldocruzia filiformis specimens were successfully amplified with JB3 and JB4, five primers without non-specific PCR-products. The sequence lengths were of about 442 bp after contigs assembly. Alignment of the newly obtained sequences against the full mitochondrial genome of T. vitrinus (GeneBank № NC_013807.1) confirmed that Oswaldocruzia filiformis sequences belonged to CoxI mtDNA region.
Maximum Likelihood and Bayesian inference trees were identical in terms of recognised relationships among species of Ancylostoma and Chabertia. Bootstraps of clades in ML analysis among Oswaldocruzia filiformis subclades were extremely low, and their topology disagreed between ML and BI analysis. Because of this, the relationships of representatives of O. filiformis isolates are described only after BI analysis (Fig. 6).

Discussion
In this study, we described the morphology of several isolates of the nematodes from the genus Oswaldocruzia and obtained new molecular phylogenetic data on them. Here we present an analysis of morphological characters traditionally used in the taxonomy of this genus.
The nematodes that we found belong to the Palearctic group of Oswaldocruzia species. They are characterized by «idiomorphic» spicules with three main branches («blade», «fork» and «shoe»), with the spicular «fork» divided above the level of its distal third (Ben Sliman et al., 1996a). Based on type II of the caudal bursa (Durette-Desset & Chabaud, 1981), the nematodes examined by us could be assigned to any of the four species: Oswaldocruzia filiformis, O bialata Molin, 1860, O. duboisi or O. skrjabini Travassos, 1937.
Oswaldocruzia filiformis was re-described by Ben  on the basis of the structure of the synlophe, spicules, and the caudal bursa of males. On the basis of the structure of spicules and the caudal bursa of males, Travassos (1937) described Oswaldocruzia skrjabini from Zootoca vivipara. The description of this nematode is also contained in Svitin (2015Svitin ( , 2016. Oswaldocruzia duboisi was described by Ben , taking into account the structure of the synlophe and spicules. The description of this parasite is also contained in Svitin & Kuzmin (2012), Svitin (2016). The re-description of Oswaldocruzia bialata, taking into account the structure of the synlophe, is contained in Durette-Desset et al. (1993). The description of this nematode is also contained in Svitin (2016).
The body sizes of Oswaldocruzia nematodes taken from various species of amphibians and reptiles in our study were different (Table 3, Table 4, Table 5, Table 6). The mean body size of the Oswaldocruzia males collected by us was bigger than of males of Oswaldocruzia filiformis, O. bialata, O. duboisi and O. skrjabini (Ben Slimane et al., 1993;Durette-Desset et al., 1993;Svitin & Kuzmin, 2012;Svitin, 2015Svitin, , 2016. The males collected from Lacerta agilis and Zootoca vivipara were an exception. Their mean size was about the same as that of Oswaldocruzia filiformis in Ben  and Svitin (2016) (Table 3, Table 5, Table 7).
In our study, the mean body size of the Oswaldocruzia females was also bigger than of females of the mentioned above four species in comparison. Oswaldocruzia females from lizards and snakes were an exception. Their mean size was slightly smaller than of Oswaldocruzia filiformis Svitin, 2016) (Table 4, Table 6, Table 8). Intraand interpopulational variability of nematodes from vertebrates and, in particular, amphibians was also noted by Tarasovskaya (2009, 2011), Tarasovskaya & Pashkevich (2011, Kirillova et al. (2012), Kirillov & Kirillova (2015), who showed that the body size of nematodes varied under the influence of such factors as sex, age, phenotype and host species, number of parasites in the host, seasonal changes and others.   Oswaldocruzia duboisi (Svitin & Kuzmin, 2012) Oswaldocruzia bialata (Svitin, 2016) Oswaldocruzia skrjabini (Svitin, 2015)     Oswaldocruzia duboisi (Svitin & Kuzmin, 2012) Oswaldocruzia bialata (Svitin, 2016) Oswaldocruzia skrjabini (Svitin, 2015)  According to the structure of spicules, the nematodes found in amphibians and reptiles could only be assigned to Oswaldocruzia filiformis. In our nematodes, the spicular «blade» is divided at the end into three branches, while the «fork» and the «shoe» did not have extra branches. On the contrary, in Oswaldocruzia bialata and O. duboisi, the blade is sharp at the end and not divided into branches (Ben Durette-Desset et al., 1993;Svitin & Kuzmin, 2012;Svitin, 2016). Oswaldocruzia skrjabini has extra branches on the «fork» and the «shoe», and the blade is divided at the end into four branches (Svitin, 2015). In our study, all nematodes have an additional branch on each ray 10, which is also present in Oswaldocruzia skrjabini, but not found in O. filiformis, O. bialata and O. duboisi.
There are differences in the number of crests at the mid-body of the nematodes we studied with the existing descriptions of the four species mentioned above (Table 3, Table 4, Table 5, Table 6, Table 7,  Table 8). So, in our study, the number of crests in Oswaldocruzia males varied from 34 to 47, while in females it varied from 39 to 77. For comparison, both males and females of Oswaldocruzia bialata have about 50 ridges in the middle of the body Svitin, 2016 filiformis has about 40 ridges (Ben Svitin, 2016) (Fig. 3, Fig. 4, Fig. 5, Table 3,  Table 4, Table 5, Table 6, Table 7, Table 8). Oswaldocruzia bialata is characterised by the absence of crests on the dorsal and ventral body sides at the oesophagus level . In our study, nematodes always had crests at the oesophagus level. In our study, the number of crests varied in nematodes of the same sex, even those taken from one host individual. The number of crests in young and adult nematodes of the same sex also differed.
The degree of development and the shape of the lateral alae were also variable, both in nematodes from hosts belonging to different species and in different nematodes recovered from one and the same host individual (Fig. 3, Fig. 4, Fig. 5). So, in one Bufo bufo individual, we found one Oswaldocruzia female with small narrow cervical alae formed by three slightly increased crests (dorsal and ventral crests and a smaller central crest between them) and two males with more developed ventral crest in lateral alae at the level of the middle of the oesophagus (Fig. 3G,H,I). Moreover, the structure of the spicules of these Oswaldocruzia males was identical with that of the nematode males from Pelophylax ridibundus and Natrix natrix (Fig. 2E, Fig. 3D, Fig. 5C).
It should be noted that there are little data in the literature on the variability of the number of crests, the shape and the degree of development of lateral alae in Oswaldocruzia spp. depending on the population structure of the parasites and the characteristics of their hosts (e.g., sex, age, phenotype). Only Ben Slimane & Durette-Desset (1996a,b) Ben , Durette-Desset et al. (2006) and Guerrero (2013) indicated differences in the number of crests in nematodes of different sexes.
Thus, in our material, we observed a broad morphological variability of nematodes both from different host species and from one host individual. Variability was observed in the size of the nematode body and individual organs, in the shape and size of the cephalic vesicle, in the shape and development degree of the lateral alae, in the number of crests on the transverse sections at the mid-body level and in the shape of rays 9 and 10 of the caudal bursa. Variation of all these characters in nematodes from various host species can be attributed to the host-induced morphological variability as has been shown on other spe-as has been shown on other species of helminths (Amin, 1975;Roytman & Kazakov, 1977;Machado-Silva et al., 1994;Anikieva, 2004;Kirillova et al., 2012;Nadler et al., 2013;Catalano et al., 2015). Variation of features in one host individual may be phenotypic (Anikieva, 2005(Anikieva, , 2008Kirillov & Kirillova, 2010;Viney & Diaz, 2012).
The nematodes collected in our study could not be identified with certainty as any of the Oswaldocruzia species, whose descriptions are available in the recent literature. However, they corresponded well to the morphological description of Oswaldocruzia filiformis (= O. goezei Skrjabin & Schulz, 1952) in «older» reviews by Skrjabin et al. (1954), Sharpilo (1976) and Ryzhikov et al. (1980), who did not take into account the structure of the synlophe.
The molecular phylogenetic analysis also showed that Oswaldocruzia nematodes collected in this study from different species of amphibians and reptiles in European Russia belonged to the species Oswaldocruzia filiformis s.l. Thus, our data contradict the opinion that several species of this genus parasitise different host species in the Western Palearctic , 1995Svitin, 2015Svitin, , 2016Svitin, , 2017.
The phylogenetic distance between nematode individuals collected in geographically distant regions was approximately the same as that between parasites from geographically close habitats. Unfortunately, there are still little data on the molecular phylogeny based on the CoxI mtDNA gene for this nematode genus and it is thus impossible to make extensive conclusions.

Conclusions
Our morphological and molecular phylogenetic data indicated that amphibians and reptiles in European Russia harbour only one species of the genus Oswaldocruzia, O. filiformis s.l. The analy- The analysis of the morphological features of Oswaldocruzia nematodes both from a single host species and from different host species showed a broad mor-a broad mor-broad mor-broad mor-mor-morphological variability. They are in conflict with the literature records, according to which most Oswaldocruzia spp. noted in the Western Palearctic are specific of amphibian and reptilian species, in which they are found. The results of our study highlight the necessity to study the diversity of morphologically similar Oswaldocruzia spp. from the Western Palearctic by genetic methods.