An Overview of the Helminths of Moor Frog Rana arvalis Nilsson, 1842 (Amphibia: Anura) in the Volga Basin

: This is the ﬁrst review of the helminth fauna of the moor frog Rana arvalis Nilsson, 1842 from the Volga river basin (Russia). The article summarizes the authors’ and literature data on the helminthic fauna of this species. The method of complete helminthological dissection was used. Thirthy-eight helminth species were recorded from three classes: Cestoda (1), Trematoda (28), and Chromadorea (9). Nine helminth species are new to the moor frog in Russia: trematodes Gorgodera varsoviensis Sinitzin, 1905, Strigea falconis Szidat, 1928, larvae, Neodiplostomum spathoides Dubois, 1937, larvae, Tylodelphys excavata (Rudolphi, 1803), larvae, Pharyngostomum cordatum (Diesing, 1850), larvae, Astiotrema monticelli Stossich, 1904, larvae and Encyclometra colubrimurorum (Rudolphi, 1819), larvae, nematodes Strongyloides spiralis Grabda-Kazubska, 1978 and Icosiella neglecta (Diesing, 1851). The cestode Spirometra erinacei (Rudolphi, 1918), larvae were observed of this amphibian species in the Volga basin for the ﬁrst time. The nematodes Rhabdias bufonis , Oswaldocruzia ﬁliformis , Cosmocerca ornata and the trematode Haplometra cylindracea form the core of the helminth fauna of the moor frog. Information on species of helminths includes systematic position, localization, areas of detection, type and scheme of life cycle, geographical distribution, and degree of speciﬁcity to host amphibians.


Introduction
In recent decades, the study of amphibian parasites has become more relevant.This is due to the crisis that this group of vertebrates is experiencing.Amphibian populations are sharply declining around the world [1][2][3].About a third (32.4%) of the world's amphibian species are on the verge of extinction [4].The disappearance, degradation, and pollution of habitats as a result of anthropogenic activities are the main factors contributing to the reduction of the range and abundance of many species [5][6][7][8].There are constant reports of amphibian diseases caused by viruses [9,10], bacteria [11], mycos [12][13][14], and helminths.
The pathogenic effect of helminthic invasion on amphibians has a wide range of manifestations: from cellular changes to inhibition of behavioral reactions.Trematodes play an important role in these processes.For example, multiple clusters of Holostephanus volgensis cysts during the metamorphosis of moor frog larvae cause the appearance of skeletal anomalies [15].Cercariae of the genus Ribeiroia lead to limb malformations in tadpoles of North American amphibians [16][17][18].The formation of deviant forms of skeletal elements significantly reduces the survival rate of the young generation of amphibians and facilitates their consumption by predators.Another type of pathology is a violation of the functioning of the internal organs of the hosts, leading to their death.For example, multiple renal invasion by Echinostoma and Echinoparyphium metacercariae contributes to the development of edema and renal failure [18].The concentration of Pleurogenes hepaticola in the bile ducts of the green frog causes liver cirrhosis and enlargement and deformation of bile-ducts and gall-bladder [19,20].Metacercariae Codonocephalus urnigerus, when intensely infested, causes pathological changes in the gonads (ovarian dystrophy, testicular tumors) in the moor frog and lead to parasitic castration of the hosts.In infected individuals, food reflexes, sexual instincts, territorial and migration behavior are suppressed.Such individuals do not participate in the breeding process, do not respond to threats, are inactive, and become easy prey for predators [21].Localization of Brandesia turgida in the duodenum of Pelophylax ridibundus is accompanied by granulomatous inflammation of its serous membrane with the development of a clinical picture of histological and cytological pathologies [22].
The moor frog, Rana arvalis Nilsson, 1842 is a widely distributed Eurasian species ranging from the Eastern France in the west to the Baikal Lake in the east and from Kola Peninsula in the north to the southern part of the Pannonian Basin [23].This species lives in a wide variety of natural and anthropogenic biotopes.It is found in various types of forests, swamps, meadows, and pastures, in ravines, near streams and rivers, and it inhabits rural areas and suburban parks [24][25][26][27].The moor frog is resistant to freezing, which is the reason for its wide distribution [28].
The Volga is the longest river in Europe.Its length is 3690 km and the basin area is about 1.4 million km 2 .It occupies 33% of the territory of European Russia and almost 13% of Europe.It flows through different types of biomes from taiga in the North to semi-desert in the South [55].The Volga basin includes total or part of the territory of 38 regions of Russia [56], which are home to 13 species of tailless and tailed amphibians, including 2 species of brown frogs (genus Rana): Rana temporaria Linnaeus, 1758 and R. arvalis [57].
In contrast to other frogs, whose helminthic fauna was studied in detail earlier in the Volga basin [58][59][60][61], data on helminths of moor frog remain scattered.Prior to this research, there was no summary on the helminths of R. arvalis, which inhabit the Volga river basin.Such a paper is needed due to the abundance of such information, changes in the systematics of helminths, and the long-term nature of the observations.The first information on this issue was obtained in the second half of the last century in the Republics of Bashkortostan [62,63], Tatarstan [64,65], and the Nizhny Novgorod (former Gorky region) region [66][67][68][69].Some of these studies are presented in the monograph of K. M. Ryzhikov et al. [46], which was then included in the European reports [52,53].In the last 15 years, new information has become available from the Vologda, Ivanovo, Samara and Saratov regions, the Republic of Mordovia, and data from the Republic of Bashkortostan has been updated.This publication is the first review of the R. arvalis helminth fauna from populations in the Volga river basin (Russia).
Study area: This study is based on the results of our own research, as well as on the data of other authors for the period from 1974 to 2020.The material for the study was personal fees and information about helminths from 990 specimens of moor frogs from eight regions of the Volga basin: Vologda, Ivanovo, Nizhny Novgorod, Samara and Saratov regions, the Republics of Mordovia, Tatarstan, and Bashkortostan (Figure 1).The method of complete helminthological dissection was used.Coll and processing of helminthological material were performed by standard m atodes and cestodes were immobilized by heating, fixed with 70% ethanol u and stained with alum carmine and encased in Canadian balsam.A dime solution was used to enlighten flatworms.The nematodes were straighten fixed with Barbagallo liquid (3% formalin solution in NaCl saline).Nemato ied both on temporary vouchers with enlightenment in lactic acid, and on with the conclusion in glycerol-gelatin.Own vouchers of worms and parti hosts are stored in Parasitological and Herpetological collections of the In ogy of Volga Basin of RAS, Samara Federal Research center Russian Acade Togliatti, Russia.The species of helminths was identified according to th of K. M. Ryzhikov et al. [46] and V. E. Sudarikov et al. [70].The systematic is based on modern data on trematodes [71][72][73][74] and nematodes [75].For intermediate hosts of trematodes and cestodes, data was used from the w Europaea" (http://www.faunaeur.org)[76].
There is an annotated list of species of the moor frog helminths inhab and transformed biocoenoses of the Volga river basin (Appendix A).It in tematic position and localization of the parasite, the areas of detection, an distribution.The method of complete helminthological dissection was used.Collection, fixation and processing of helminthological material were performed by standard methods.Trematodes and cestodes were immobilized by heating, fixed with 70% ethanol under pressure, and stained with alum carmine and encased in Canadian balsam.A dimethyl phthalate solution was used to enlighten flatworms.The nematodes were straightened by heat and fixed with Barbagallo liquid (3% formalin solution in NaCl saline).Nematodes were studied both on temporary vouchers with enlightenment in lactic acid, and on total vouchers with the conclusion in glycerol-gelatin.Own vouchers of worms and partially amphibian hosts are stored in Parasitological and Herpetological collections of the Institute of Ecology of Volga Basin of RAS, Samara Federal Research center Russian Academy of Sciences, Togliatti, Russia.The species of helminths was identified according to the determinants of K. M. Ryzhikov et al. [46] and V. E. Sudarikov et al. [70].The systematics of helminths is based on modern data on trematodes [71][72][73][74] and nematodes [75].For describing the intermediate hosts of trematodes and cestodes, data was used from the website "Fauna Europaea" (http://www.faunaeur.org(accesssed on 3 February 2021)) [76].
There is an annotated list of species of the moor frog helminths inhabiting protected and transformed biocoenoses of the Volga river basin (Appendix A).It includes the systematic position and localization of the parasite, the areas of detection, and geographical distribution.

Results
The helminth fauna of the moor frog R. arvalis in the Volga river basin includes 38 species of helminths.Systematically, they belong to 30 genera, 16 families, 7 orders, and 3 classes: Cestoda-1, Trematoda-28, and Chromadorea-9 (Table 1 According to the degree of hostal specificity, all species of helminths are divided into 3 groups.Most of them (23 species) are polyhostal parasites of tailless amphibians.A minority (14) are oligohostal for frogs of the family Ranidae.Another nematode species, Cosmocerca commutata (Diesing, 1851), is a monohostal parasite of the green toad (Bufotes viridis Laurenti, 1768).
The moor frog serves as the definitive host for most helminth species (22), which parasitize exclusively at the adult stage of development (Table 1).For some cestode (1) and trematode (10) species that parasitize at the larval stage, the amphibian is an intermediate, intercalary (mesocercarial), additional (metacercarial), and/or paratenic (metacercarial) host.The remaining 5 species of trematodes (G.vitelliloba, H. cylindracea, D. rastellus, O. ranae) and nematode (C.commutata) combine the larval and adult stages of development in the amphibian body.In relation to such helminths, the moor frog plays the role of an amphixenic host.Brief information on the biology of helminths with the indication of host categories is given in Table 2.
The largest number of helminth species has been recorded in the moor frog from the Republic of Mordovia (26 species), the Samara region (24), and the Republic of Bashkortostan (22); the smallest number has been recorded in the Republic of Tatarstan (9), Vologda (7), and Ivanovo (6) regions.The same indicator for this host in the Nizhny Novgorod (16) and Saratov (10) regions is characterized by intermediate values (Table 1).The reasons for the differences are usually complex: from the different number of amphibians studied in a particular region to the biotopic differences in their habitats, types of biomes, geographical location, climate, and the degree of anthropogenic impact.It is difficult to carry out statistical processing of data that were obtained by different authors at different times and in different seasons.However, even on the basis of the mentioned information, conclusions can be drawn.It is not only the intensive study of helminth fauna that has affected the list of species in recent years.For example, the number of materials studied was similar in the Republic of Mordovia and the Republic of Tatarstan.However, the species richness of helminths differed significantly.We think that this depends on the geographical and climatological characteristics of the range of R. arvalis.The Republic of Tatarstan has a more continental climate, which influences the activity of amphibians.
The composition of helminths of the moor frog differs in certain regions of the Volga basin.Comparative analysis shows that only three species out of 38 were found in all samples: the nematodes Rh. bufonis, O. filiformis, and C. ornata.Among trematodes, the most widespread species is H. cylindracea, which has been recorded in seven regions.This group of helminth species (4) forms the core of the helminth fauna of the moor frog in the Volga river basin.Largest group of species (26) are common parasites of this host, occurring sporadically: in five (3 species), four (6), three (9), and two (8) regions.The last 8 species of cestodes (S. erinacei, larvae), trematodes (H.ovocaudatus, G. varsoviensis, B. turgida, T. excavata, larvae, Ph. cordatum, larvae) and nematodes (S. spiralis and I. neglecta), belong to the group of rare or accidental parasites of the moor frog and have a strict local confinement (Table 1).

Discussion
The biology and ecology of the moor frog is characterized by a number of life-history traits: (1) active terrestrial lifestyle in wet forest, forest-steppe, and even steppe stations; (2) feeding on terrestrial invertebrates (oligochaetes, mollusks, insects, millipedes, arachnids); (3) visiting ponds and lakes during spawning; (4) "mating fasting".The range of R. arvalis in the Volga basin is extensive and the habitats are extremely diverse [126].
These factors determine the species composition, structure and community of the helminth fauna in this frog, distinguishing 3 groups of parasite species.The first group (18 species) includes adult biohelminths (trematodes).They are characterized by a variety of species, but usually have a low infestation level.The reason is feeding on terrestrial prey, the seasonal visit of water bodies for reproduction and the "mating fasting" of the host.The second group (8) includes adult geohelminths (nematodes).With a smaller number of species, they have a wide distribution, frequent occurrence and a high level of invasion, which is influenced by the lifestyle of the moor frog.The third group (11) includes larval biohelminths (trematodes, cestodes).They occur sporadically and with different infestations depending on the presence of predators of a higher trophic level and the degree of habitat degradation.
The moor frog plays an important biocoenotic role as prey of a number of predators: snakes, owls, crows and diurnal birds of prey, canine and feline mammals.This is evidenced by the presence of larval stages of helminths (cestodes and trematodes).They circulate through trophic levels and complete their development in the body of representatives of these groups of vertebrates.In addition to the definitive hosts, this amphibian species has the ability to transmit mesocercariae and metacercariae of individual trematode species (families Strigeidae, Diplostomidae) a vast number of their reservoir hosts: green frogs, vipers and lizards, ducks, gulls and chickens, rodents, martens, insectivorous and canine mammals, wild boars.These facts indicate the significant role of the moor frog in the circulation of parasites in terrestrial and near-water natural biocoenoses of the Volga river basin.Changes in natural communities can negatively influence the population of R. arvalis.A decrease in the number of this species will lead to changes in the circulation of helminths along trophic chains within the ecosystem.In this regard, it is necessary to monitor the impact of climate change on the helminth fauna of R. arvalis in different parts of its range.Funding: The research for this paper had no direct external funding to the authors.
Institutional Review Board Statement: All applicable institutional, national, and international guidelines for the care and use of animals were followed.Directive of the European Parliament and the Council of the European Union of 22 September 2010 "On the Protection of Animals Used for Scientific Purposes" (EU directive 2010/63/EU).
Areas of detection: Samara region [133].It was discovered in the moor frog of the fauna of Russia and the Volga basin for the first time.
Areas of detection: Samara region [128,129,131,133,135].It was discovered in the moor frog of the fauna of Russia and the Volga basin for the first time.
Encyclometra colubrimurorum (Rudolphi, 1819), larvae Localization: body cavity, serous coat of internal organs (liver, spleen, ovary), mesentery.Areas of detection: Samara [132,133,135] and Saratov [131,135] regions.It was discovered in the moor frog of the fauna of Russia and the Volga basin for the first time.

Figure 1 .
Figure 1.Location of the Volga basin.

Figure 1 .
Figure 1.Location of the Volga basin.

Author Contributions:
Conceptualization, I.V.C.; methodology, I.V.C. and A.B.R.; validation, I.V.C. and A.B.R.; formal analysis, I.V.C. and A.B.R.; investigation, I.V.C.; data curation, I.V.C. and A.B.R.; writing-original draft preparation, I.V.C.; writing-review and editing, I.V.C. and A.B.R.All authors have read and agreed to the published version of the manuscript.

Table 2 .
Life cycles of helminths of a moor frog Rana arvalis.