Oswaldocruzia ukrainae (Nematoda: Molineidae)—A Parasite of European Green Toad Bufotes viridis: Morphological and Molecular Data

Simple Summary Roundworms of the genus Oswaldocruzia are common parasites of the small intestine of amphibians and reptiles. Our recent study revealed that only one species, Oswaldocruzia filiformis, parasitizes anurans, lizards and snakes in European Russia. In this work we studied nematodes of the genus Oswaldocruzia in the European green toads from the Middle Volga region using traditional morphological and molecular genetic methods. Descriptions, original drawings, and data on molecular phylogenetic analysis for studied nematodes were produced. The results of our study showed that green toads are parasitized by two Oswaldocruzia species, Oswaldocruzia ukrainae and O. filiformis. We found broad variability in morphology of the studied nematodes, which can lead to inaccurate identification of closely related species of Oswaldocruzia. The data obtained in our study should contribute to the knowledge of Oswaldocruzia nematodes and host-parasite associations in general. Abstract Nematodes of the genus Oswaldocruzia are common parasites of the small intestine of amphibians and reptiles. Our recent molecular analysis of Oswaldocruzia nematodes revealed that only Oswaldocruzia filiformis, which possesses high morphological variability, parasitizes amphibians and reptiles in European Russia. Here we present the study of Oswaldocruzia nematodes from the European green toad Bufotes viridis (Anura, Bufonidae) collected at different localities of the Middle Volga region in 2018–2022. We analyzed the morphological characteristics of the Oswaldocruzia spp. taxonomy together with novel molecular phylogenetic data. The data on phylogenetic analysis (based on partial CoxI mtDNA gene sequences) showed that Bufotes viridis is parasitized by two Oswaldocruzia species, the host-specific parasite Oswaldocruzia ukrainae and species generalist Oswaldocruzia filiformis. Broad morphological variability was revealed in O. ukrainae nematodes both from the same host specimen and from various toad individuals from different localities. Our results highlight the need for further biodiversity research of morphologically similar Oswaldocruzia species from amphibians and reptiles in the Western Palearctic using molecular genetic methods.


Introduction
The distribution range of the European green toad, Bufotes viridis Laurenti, 1768 (Amphibia: Bufonidae), runs from eastern France to the Balkans and European Russia. Previously, this toad species also inhabited the Middle East and Central Asia, reaching western China and northwestern India, as well as in the south to North Africa. Nowadays, toad populations inhabiting North Africa, the Balearic Islands and Asia are considered separate species [1,2].
Nematodes from the genus Oswaldocruzia Travassos, 1917 are one of the most common parasites of B. viridis. These nematodes parasitize the small intestine of amphibians and reptiles, mainly in anurans. This genus includes about 90 species distributed worldwide .
In Russia, the nematode O. ukrainae was previously identified in B. viridis only in the Stavropol, Volgograd and Astrakhan regions [3,38,39]. Oswaldocruzia ukrainae has also been recorded in toads from the Poltava region of Ukraine [33], Uzbekistan [13,63] and Tunisia [4]. In addition, O. iwanitzkyi, a new species for Russia, was revealed in B. viridis from the Astrakhan region [38,39]. This species has previously been recorded only in green toads of Ukraine [64] and Slovakia [66]. During the helminthological study of toads in the type area, Svitin [33] found only O. ukrainae in B. viridis. Therefore, the validity of O. iwanitzkyi requires clarification.
The species O. ukrainae is a parasite specific to B. viridis and has not been recorded in other amphibian species. On the contrary, the nematode O. filiformis is a species generalist parasitizing most species of amphibians in the Palaearctic. This species greatly exceeds all other nematodes of amphibians in terms of the number of hosts. Twelve amphibian species have been registered as hosts for O. filiformis in Russia and neighboring countries [3,52].
We have previously identified Oswaldocruzia nematodes from various amphibian species in the Middle Volga region as O. filiformis [35,55,[67][68][69]. Our recent molecular and morphological analysis of Oswaldocruzia nematodes revealed that only one species, O. filiformis, parasitizes amphibians and reptiles in European Russia [40]. Unfortunately, that study did not include Oswaldocruzia specimens parasitizing the European green toads, B. viridis.
The purpose of this study was species identification of Oswaldocruzia nematodes parasitizing B. viridis in European Russia using morphological and molecular phylogenetic analysis. We also compared the newly obtained results with our previous data.

Nematode Collection and Examination
Nematodes of the genus Oswaldocruzia were collected from the small intestine of B. viridis at six localities in the Middle Volga region (European Russia) in 2018-2022: vicinity of Spassk city (Penza region, 53 • Figure 1 and Table 1. (53°12′27″ N, 50°9′18″ E) and Oktyabrskiy township (Samara region, 53°25′33″ N, 52°3′48″ E). Data on the geographic origin of the examined Oswaldocruzia nematodes are presented in Figure 1 and Table 1.  No amphibians were intentionally killed for this research. In this work, we studied only road-killed toads. Several dead amphibians were kindly provided by the rural residents. The necropsy was performed on toads approximately 1-8 h after their death. Only alive motile adult nematodes were collected for further investigation. Some nematodes were collected from toads and preserved in 96% ethanol for further molecular phylogenetic analysis. For the morphological analysis, the nematodes were killed by heating in water, then parasites were cleared in lactic acid. In this work we studied and measured 59 Oswaldocruzia nematodes, including 26 males and 33 females.
Microscopic examination, morphological measurements and drawings of the nematodes were performed using MBI-9 light microscope, drawing tube RA-7 and the Levenhuk M500 BASE Digital Camera. We made apical and transverse nematode sections manually using a razor blade. The synlophe and spicules were described in accordance with Durette-Desset [72]. The nomenclature of the male caudal bursa is specified following Durette-Desset and Chabaud [73]. The studied morphological characteristics were the shape of cephalic vesicle, the shape of lateral alae at mid-esophagus level, the number of crests in the level of midbody, the shape and structure of male caudal bursa, the shape of male spicules, the shape and structure of the dorsal ray of bursa. We also examined such morphometric features as body length, width at mid-body level, length of esophagus, length and width of cephalic vesicle, tail length, spicule length, egg length and width, and distances from the anterior end of the body to the vulva, excretory pore and nerve ring.

DNA Extraction, Amplification, Sequencing, and Phylogenetic Analysis
In order to obtain partial CoxI mtDNA sequences, seven specimens of ethanol-fixed Oswaldocruzia were dried at 37 • C in a dry block heater for 1 h. The specimens were then transferred to clear 500 µL tubes with a mixture of 48 µL 0.1% Chelex-100 and 2 µL Proteinase K (concentration 10 mg/mL) and incubated for 2 h at 55 • C and 25 min at 95 • C to stop proteinase activity. After that, the water solution of the total DNA was transferred to a sterile 500 µL tube and frozen at −20 • C. The amplification primer pair was JB3 (5 -TTT TTT GGG CAT CCT GAG GTT TAT-3 ) and JB4,5 (5 -TAA AGA AAG AAC ATA ATG AAA ATG-3 ) [74]. PCR was performed in 25 µL of reaction mix per PCR-tube in a BioRad C1000 thermal cycler. The following parameters were used: initial denaturation (3 min at 95 • C) followed by 35 cycles of 20 s at 95 • C, 20 s at 53 • C and 40 s at 72 • C, with 5 min at 72 • C for final extension [40]. Amplicons were directly sequenced using ABI-PRISM 3500xl sequencer (Life Technologies, Carlsbad, Massachusetts, USA) with the same primers. The sequences were mounted in general alignment with other nematode species, which were downloaded from GenBank NCBI using a custom R script [75,76] (Table 1). The sequences were automatically aligned using Muscle algorithm [77] as implemented in SeaView 4.0 [78]; the alignment was then trimmed manually. The phylogenetic analysis was performed using the maximum likelihood method at Cipres portal [79] with GTR+G+I model and a non-parametric bootstrap with 1000 pseudoreplicates. Bayesian analysis was performed with MrBayes on XSEDE 3.2.7a (GTR+G+I model). The model for phylogenetic analysis was chosen with MrModeltest 2.3 [80]. Trees were run as two separate chains (default heating parameters) for 15,000,000 generations. The quality of the chains was estimated using built-in MrBayes tools and, additionally, using the Tracer 1.6 package [81]. Based on the estimates, the first 25,000 generations were discarded for burn-in.
The p-distances were calculated using MEGA11 software [82] with standard parameters. Results of p-distance estimation were used as input data for 'ComplexHeatmap' library in R programming language [76,83].

Results
We found the nematodes O. ukrainae during a helminthological study of B. viridis from various localities of the Middle Volga region ( Figure 1). These specimens were identified in toads from the Republic Mordovia and Samara region. At the same time, during the study of one individual of B. viridis from the vicinity of the Spassk city (Penza region), one male of O. filiformis was revealed.

Morphology of Oswaldocruzia ukrainae
The body of the nematode is thin and elongated, with maximum width at mid-length. The anterior body end has a cephalic vesicle that is variable in shape (Figures 2b and 3h).
Shape of vesicle is variable even in specimens of O. ukrainae from one individual toad. The cuticle forms continuous longitudinal ridges beginning behind the cephalic vesicle and running along the whole nematode body, forming a synlophe, which is symmetrical. Lateral alae are absent ( Figure 3). The number of crests at mid-length of the body varies depending on sex of nematodes (Figures 2 and 3, Tables 2 and 3). Ridges are not continuous from the anterior end of body to the tail. They can end in one area, and then appear in another body part. The anterior end of the body is rounded. The triangular oral opening is surrounded by four large cephalic papillae and six external-labial papillae, of which the lateral papillae are close to small, not always distinguishable amphids (Figure 3). The esophagus is club-shaped, thin, cylindrical in the anterior part and wide in the posterior part. The posterior end of the esophagus is rounded with a posterior bulb (Figures 2 and 3). Excretory pore position varies within the posterior third of the esophagus. The nerve ring surrounding the esophagus in the middle part is slightly closer to its posterior third (Figures 2 and 3).          Male. The body ends with a wide caudal bursa that is symmetrical and three-lobed (two lateral and one dorsal). According to the classification of Durette-Desset and Chabaud [73], it belongs to type II. Ray 9 in male bursa always have a more or less S-shaped bend. Ray 10 always has extra branches of variable size (Figure 2). The genital cone is well developed, with two papillae (Figure 2). Gubernaculum is absent. Spicules are approximately equal and surrounded by a thin membrane. According to Durette-Desset [72], spicules are nonidiomorphic, with three branches: the internal branch is wide in the anterior part and narrows towards the posterior sharp end (Figure 2). The central branch is the longest, is rounded at the end, and has no extra offshoots. The external branch is narrow, sharp at the end; at the level of the last third it has a small offshoot (Figure 2). Measurements are presented in Table 2.
Female. The body is always larger than in males, tapering to the posterior end. The tail has a thin tip (Figure 3). The female reproductive system, like in all Oswaldocruzia species, is amphidelphic. Vulva is wide, opening postequatorially, behind the body at mid-length. The muscular vagina passes into paired ovejectors, which have powerful sphincters (Figure 3). The anterior ovary forms numerous loops in the anterior body part. The ovary reaches the level of esophagus and returns. The posterior ovary extends to the body end, strongly bends and turns forward slightly twisting, going beyond the level of the vulva. The uterus of adult females is filled with eggs. All eggs in vulva, ovejector and uterus were found at morula stage. Measurements of the main characteristics are presented in Table 3.

Morphology of Oswaldocruzia filiformis Male
The body is thin and elongated. The cephalic vesicle presents on the anterior body end (Figure 4). The cuticle forms longitudinal ridges starting behind the cephalic vesicle and running along the whole body. The anterior end of the body is rounded. The triangular oral opening has four large cephalic papillae and six externo-labial papillae. The esophagus is thin, cylindrical in the anterior part and wide in the posterior part. The posterior end of the esophagus forms a bulb (Figure 4). The excretory pore is located at the level of the posterior third of the esophagus. The esophagus is surrounded by a nerve ring at the level of its middle part. Synlophe is symmetrical. On transverse sections, at the level of the second third of the esophagus, small cervical alae are clearly visible, formed by three slightly enlarged crests (Figure 4). Lateral alae consist of three ridges, with a more developed ventral crest. Two much smaller ridges (dorsal and central) are directly above the large ventral crest (Figure 4). At the level of the anterior part of the intestine, the lateral alae transform into simple ridges. The number of crests at mid-length of the body is 40 (Figure 4). A wide caudal bursa is at the end of the body. The caudal bursa is three-lobed, symmetrical and belongs to type II [73]. Ray 9 is in the form of an S-shaped bend. Extra branches on ray 10 are not expressed (Figure 4). The genital cone is well developed and has two papillae ( Figure 4). According to Durette-Desset [72], spicules are idiomorphic, approximately equal and surrounded by a thin membrane. Each spicule has three branches, namely "blade", "fork" and "shoe". The end of The excretory pore is located at the level of the posterior third of the esophagus. The esophagus is surrounded by a nerve ring at the level of its middle part. Synlophe is symmetrical. On transverse sections, at the level of the second third of the esophagus, small cervical alae are clearly visible, formed by three slightly enlarged crests (Figure 4). Lateral alae consist of three ridges, with a more developed ventral crest. Two much smaller ridges (dorsal and central) are directly above the large ventral crest (Figure 4). At the level of the anterior part of the intestine, the lateral alae transform into simple ridges. The number of crests at mid-length of the body is 40 (Figure 4). A wide caudal bursa is at the end of the body. The caudal bursa is three-lobed, symmetrical and belongs to type II [73]. Ray 9 is in the form of an S-shaped bend. Extra branches on ray 10 are not expressed (Figure 4). The genital cone is well developed and has two papillae ( Figure 4). According to Durette-Desset [72], spicules are idiomorphic, approximately equal and surrounded by a thin membrane. Each spicule has three branches, namely "blade", "fork" and "shoe". The end of the blade is visually divided into two branches, as well as distal parts of branches that visually split into two narrow offshoots. In fact, the branches do not split, but are tightly pressed against each other. The fork is divided into two branches approximately at mid-length. The shoe has a thin branch at mid-length and is slightly bent at its distal end (Figure 4). Measurements are presented in Table 4.

Molecular Phylogenetic Analysis
According to the results of molecular phylogenetic analysis, two clusters of specimens with almost full support are clearly distinguishable ( Figure 5). One cluster includes specimens of O. filiformis, and the other is a clade of O. ukrainae. One of the newly obtained sequences, belonging to O. filiformis, clustered together with all previously generated ones, although this specimen was collected from B. viridis, a typical host for O. ukrainae.
The presence of two clusters is also confirmed by the results of p-distance estimation. On average, the p-distance among specimens of the same species is about 0.01, but approximately 0.11 between O. filiformis and O. ukrainae ( Figure 6). As in phylogenetic reconstruction, a single specimen of O. filiformis collected from B. viridis is clustered together with other representatives of this species from different hosts. One cluster includes specimens of O. filiformis, and the other is a clade of O. ukrainae. One of the newly obtained sequences, belonging to O. filiformis, clustered together with all previously generated ones, although this specimen was collected from B. viridis, a typical host for O. ukrainae.
The presence of two clusters is also confirmed by the results of p-distance estimation. On average, the p-distance among specimens of the same species is about 0.01, but approximately 0.11 between O. filiformis and O. ukrainae ( Figure 6). As in phylogenetic reconstruction, a single specimen of O. filiformis collected from B. viridis is clustered together with other representatives of this species from different hosts.

Discussion
Here we present a morphological description of two species of Oswaldocruzia nematodes of the European green toad Bufotes viridis inhabiting the Middle Volga region (European Russia) and new molecular phylogenetic data for these species. The combined use of both molecular and morphological methods allowed the reliable identification of Oswaldocruzia spp.
Morphological and molecular data obtained in our work showed that Bufotes viridis in European Russia is parasitized by two species of the Oswaldocruzia genus, O. ukrainae and O. filiformis. Oswaldocruzia ukrainae is the only species of the genus in the Western Palearctic with non-idiomorphic spicules formed by three branches [33,72]. The nematode

Discussion
Here we present a morphological description of two species of Oswaldocruzia nematodes of the European green toad Bufotes viridis inhabiting the Middle Volga region (European Russia) and new molecular phylogenetic data for these species. The combined use of both molecular and morphological methods allowed the reliable identification of Oswaldocruzia spp.
Morphological and molecular data obtained in our work showed that Bufotes viridis in European Russia is parasitized by two species of the Oswaldocruzia genus, O. ukrainae and O. filiformis. Oswaldocruzia ukrainae is the only species of the genus in the Western Palearctic with non-idiomorphic spicules formed by three branches [33,72]. The nematode specimens examined in our work correspond to earlier morphological descriptions of O. ukrainae [3,33,64].
Some differences in the morphometric characteristics of the nematodes O. ukrainae were revealed, as in previous studies of O. filiformis [40][41][42]. Oswaldocruzia ukrainae specimens from different B. viridis individuals collected at various localities of the Middle Volga region and even from one host individual also exhibit broad morphometric variability. We found variability of such morphometric characteristics as the body size, cephalic vesicles and spicules, the distances from the anterior end of the body to the excretory pore, nerve ring and vulva (Tables 2 and 3). Variability in the number of ridges in the nematode mid-body was also revealed. In this study, the number of ridges in O. ukrainae males varies from 32 to 37, as well as in females from 40 to 48 (Tables 2 and 3). We determined that not all crests extend continuously from the anterior body end to the tail. Some ridges may disappear and not reach the posterior body end. At the same time, new crests may appear elsewhere in the body. This may be one of the reasons for previously noticed variations in the number of crests. Moreover, the number of crests in Oswaldocruzia nematodes varies in young and mature individuals as well as in males and females, as we have shown earlier [40][41][42]. We believe that this is directly correlated with the size of Oswaldocruzia nematodes. Therefore, the largest Oswaldocruzia females will always have the maximum number of ridges in the mid-body. This is also revealed in the case of the nematode age. Thus, young small-sized nematodes will always have fewer crests than large adults. Egg sizes remained relatively stable (Tables 2 and 3). Variability in the morphology of different O. ukrainae individuals was also revealed, as in the case of O. filiformis [40][41][42]. Thus, the shape of the head vesicle in individuals of both sexes varies greatly in nematodes from different toad individuals and from the same host specimen (Figure 2).
The spicules in O. ukrainae males were similar in structure and shape, but we noted differences in their sizes from different nematode individuals ( Table 2). The shape and structure of the caudal bursa in O. ukrainae males were constant. However, the shape and structure of the dorsal ray of the bursa, which includes the 9 and 10 rays, are varied. All O. ukrainae males had an extra offshoot on the 10th ray of different size and shape ( Figure 2). In females, the distance from the anterior end of the body to the vulva varied broadly and depended on the total length of the nematodes. Expectedly, this index was the highest in the largest nematode females (Table 3). In addition to O. ukrainae specimens, we also revealed one O. filiformis male in the examined individuals of B. viridis (Table 2 and Figure 3). Previously, we recorded O. filiformis in B. viridis from European Russia [42]. The description of specimen of O. filiformis studied in our work did not differ from the previous morphological descriptions of this species [3,33,[40][41][42]. Thus, we confirmed the possibility of parasitism of both Oswaldocruzia species in the green toad B. viridis. Previously, the parasitism of these nematode species in B. viridis was reported by Ivanitsky [64], Ryzhikov et al. [3], Vashetko and Siddikov [13], Andreev [38] and Ikromov [63].
Thus, findings of O. filiformis in B. viridis are not uncommon and are probably related to the results of syntopic habitation with other amphibian species, especially during the breeding season in semiaquatic habitats. Infection of B. viridis with the nematode O. filiformis can also occur in habitats where it lives together with the Common toad, Bufo bufo (Linnaeus, 1758).
Bufotes viridis and B. bufo are sympatric species with overlapping ranges. In European Russia, the territory of the joint habitat of both toad species coincides with the boundaries of the forest-steppe zone. Both toad species lead the same ground-burrow, twilight lifestyle. These synanthropic species inhabit the same anthropogenic environments such as parks, gardens, cellars and agricultural landscapes, and often prefer them to natural habitats [52,[84][85][86][87][88]. The similarity in lifestyles contributes to the infection of toads with common helminth species, including the nematode O. filiformis. Nematodes of this genus have a direct life cycle. Therefore, simple oral contact of toads with infective nematode larvae on a moist substrate, for example, feeding on terrestrial invertebrates, is sufficient to infect the host.
Ivanitsky [64] described another species of Oswaldocruzia that parasitizes B. viridis on the territory of Ukraine, O. fulleborni, which has not been recorded since the initial description. The main differences of the species were the following: the absence of lateral alae; synlophe consists of 30-50 crests; the "blade" branch is rounded and does not split at the end. We have previously noted varying degrees of the lateral alae development in O. filiformis from various hosts and even from the same host specimen [40][41][42]. It should be noted that the lateral alae are clearly visible only on transverse sections of nematodes, which are not presented in Ivanitsky's work [64]. We also showed that the number of ridges in O. filiformis individuals can vary depending on the sex and age of the nematodes themselves. Thus, the number of ridges in mid-body of males of O. filiformis from different amphibian species can vary from 33 to 58; and in that of females-from 39 to 78 [40][41][42]. Therefore, these two morphological characteristics cannot be used in the diagnosis of Oswaldocruzia species. According to the description and drawing of the spicules of Oswaldocruzia males by Ivanitsky [64], we did not find any differences compared to O. filiformis. The visible shape of the "blade" end of the branch varies depending on the position of the spicule. We already noted that the distal parts of the "blade" branch are divided into two thin offshoots, but it is not always clearly visible [40]. In fact, these branches are always tightly adjacent to each other. We did not observed separation or splitting of these branches on any of the spicules of O. filiformis males [40][41][42].
Judging by the description of O. fulleborni by Ivanitsky [64], Ryzhikov et al. [3] believed that there are no significant differences between O. filiformis and O. fulleborni, and considered these species to be synonymous. Ben Sliman with coauthors [7] considers this species valid due to the difference in the crest number at the level of the mid-body and the structure of the "blade" branch of the spicule. Nevertheless, we agree with the opinion of Ryzhikov et al. [3] and consider O. fulleborni to be a synonym of O. filiformis, until the opposite is proven. It should also be noted that Svitin [33] found only O. ukrainae in the green toads during the study of Oswaldocruzia from the type area of O. fulleborni and other parts of Bufotes viridis range. Due to morphological similarity with O. filiformis, the species O. ukrainae may be misidentified. Therefore, diagnosis of both species should take into account the morphological and morphometric variability. Apparently, the species O. ukrainae could be misdiagnosed as O. filiformis in most of the early studies of Oswaldocruzia nematodes from B. viridis.
When identifying Oswaldocruzia nematodes, special care should be paid to the presence/absence of lateral alae. This cuticular structure can be deformed during collection, fixation, or production of whole mounts. Therefore, it is necessary to make transverse sections of nematodes. As our studies have shown, the number of crests also cannot be a diagnostic feature, since the ranges of the number of crests in the two nematode species overlap (Tables 2-4) [40,42].
There are also relative differences in body size between the two nematode species. Adult males and females of O. ukrainae are somewhat smaller than mature O. filiformis individuals. Thus, adult O. ukrainae males were not found larger than 7.83, and the body length of the smallest males was 5.00 ( Table 2). The size of adult O. filiformis males from different species of amphibians was 6.25-13.25 [40,42]. The females of O. ukrainae in our study were 9.25-11.55 in length (Table 3), while the body sizes of adult O. filiformis females were 9.96-24.50 [40,42]. Thus, the body sizes of the nematodes should be taken into account, but not as a main feature, since they overlap. The only reliable morphological features in the diagnosis of O. ukrainae and O. filiformis are the size and structure of the male spicules (Figures 2 and 3, Tables 3 and 4) [40,42].

Conclusions
Our morphological and molecular phylogenetic data showed that the European green toad, Bufotes viridis, in the Middle Volga region is parasitized by two species of the genus Oswaldocruzia, the host-specific parasite O. ukrainae and species generalist O. filiformis. We assessed morphological characteristics for a reliable identification of two closely related Oswaldocruzia species under consideration and showed that only size and structure of male spicules can serve as the species diagnosis. The analysis of the morphological features of O. ukrainae from B. viridis both from various toad individuals and from the same host specimen reveals broad morphological and morphometric variability. In addition, high variability in O. ukrainae nematodes was revealed in the green toads from different geographical localities. The obtained data expand our knowledge of amphibian nematodes. We presented the first record of O. ukrainae parasitizing amphibians of the Middle Volga region. The results of our studies emphasize the need to confirm the validity of Oswaldocruzia species by molecular methods.