HELMINTHES OF MOUSE-LIKE RODENTS IN THE BELOGORYE STATE NATURE RESERVE (RUSSIA)

The study was conducted in 2016–2017 on three sites in the Belogorye State Nature Reserve (Russia) located in the forest-steppe zone. These sites are 1) «Les na Vorskle», upland oak ( Quercus robur ) forest; 2) «Ostrasyevy Yary», ravine oak forest, grass-meadow steppe; 3) «Yamskaya Step’», Sury valley, shrublands and forb meadow steppe, Eremkin Log valley, upland oak forest, forb meadow steppe. The small mammals investigated in this present parasitological study were caught using snap traps. These traps were established in lines of 50 or 100 traps per line. In addition, single traps and cone-shape pitfall traps (average length of 30 m) were used. The caught rodents (118 specimens) concern five species: Myodes glareolus , Microtus arvalis , Sylvaemus flavicollis , Sylvaemus uralensis , and Apodemus agrarius . The total prevalence by helminthes was 72%. About 91% of the Muridae animals were infected, while the prevalence of infestation was 55.6% among the Cricetidae specimens. We registered 20 helminth taxa belonging to three classes, six orders, and 12 families. Among the trematodes only eurixenous species were found. Among the cestodes, eurixenous species predominated. Among the nematodes, eurixenous and stenoxenous species were present in almost equal proportion. Dominant species are characterised by maximal values of prevalence of infestation. There were Hymenolepis sp. (17%) among Plathelminthes, species of Syphacia (35.6%) and Heligmosomoides (27.1%) among Nematoda, including Syphacia stroma (17%) and Heligmosomoides polygyrus (14.4%). The other helminth species registered in the present study were either small in number (with prevalence of invasion from 2% to 14%) or rare (with prevalence of invasion less than 2%). Platynosomum muris (Trematoda, Dicrocoeliidae) and Pterothominx sadovskoi (Nematoda, Capillariidae) were reported for the first time in the Central Chernozem Region of Russia.


Introduction
The biodiversity studies of certain taxonomic groups are one of the most important issues of environmental research. Environmental monitoring of particular areas without the knowledge of its flora and fauna is almost impossible. In natural ecosystems, parasitism is considered as an ecological phenomenon, the form of interspecific relationships. So the parasites having various ecological connections are a significant factor in the regulation of the functioning of the entire ecosystem (Romashova, 2004;Hudson et al., 2006).
Mouse-like rodents are one of the leading vertebrate groups in regard to the abundance and species diversity in the forest and forest-steppe ecosystems (Poulin & Morand, 2000;Orlova & Orlov, 2019). Being the consumers of the first and second orders in the food chains, the rodents play a significant role in the helminthes' circulation as intermediate and definitive hosts. They participate in the maintenance of natural focal infections and invasions (Summers et al., 2003).
The study of the helminthes of mouse-like rodents is of particular interest in Protected Ar-eas (PAs), where the zonal diversity of locally typical species and the conditions of their natural habitat are better preserved (Dobson et al., 2008;Sheykina & Zhigileva, 2018). The Belogorye State Nature Reserve is one of the main PAs in the Belgorod region (European Russia).
The paper aims to study the helminth species composition in mouse-like rodents in the Belogorye State Nature Reserve. As previously there were no helminthological studies of small mammals in the Belgorod region, it is relevant to obtain information on the helminthes species composition in the Belogorye State Nature Reserve as one of the inventory stages assessing the natural resources of the Belgorod region.

Material and Methods
To conduct the parasitological study, small mammals were caught using snap traps with treadle (Karaseva & Telitsina, 1996) during the spring -autumn of 2016-2017 on the sampling sites located in several clusters of the Belogorye State Nature Reserve. The first of them is «Les na Vorskle» (upland oak (Quercus robur L.) forest, 50.612103°N, 35.994553°E). The second site is the «Ostrasyevy Yary» (ravine oak forest and forb meadow steppe, 50.553843°N, 36.054914°E). The third site is the «Yamskaya Step'» (Sury valley: shrublands and forb meadow steppe, 51.199722°N, 37.642222°E; Eremkin Log valley: upland oak forest, forb meadow steppe, 51.177222°N, 37.650556°E).
Snap traps were arranged in lines of 50 or 100 traps each. Additionally, single snap traps were used, too. We checked the traps every 24 h. On the site «Ostrasyevy Yary», we used an additional catching method using cone-shape pitfall traps (average length 30 m) (Karaseva & Telitsina, 1996). In total, 312 snap-trap-days and 30 pitfall-trap-days were performed.
The helminthes detected during the autopsy were fixed, labeled, and delivered to the laboratory for subsequent staining and species determination (Anikanova et al., 2007). A total of 1813 helminthes were collected and analysed.
Quantitative indicators of invasion and helminth occurrence in the hosts were evaluated. We used parasite prevalence (PP), invasion in-tensity amplitude (IIA), and abundance index (AI) (Anikanova et al., 2007).

Results and Discussion
As a result, 1507 helminth specimens were determined down to species level, 302 specimens to genus level, and four specimens to family level. The helminthes have been found in 85 specimens (72%) of mouse-like rodents belonging to five species. The PP of Muridae species was 91%, exceeding almost twofold the PP of Cricetidae (55.6%) ( Table 1). We registered 20 helminth taxa belonging to three classes, six orders, and 12 families. Fourteen taxa have been identified to species level, four to genus level, and two taxa to family level. Seventeen species represent mature adults, three species larval stages ( Table 2). The PP, IIA, and AI indices of the found helminth species and their affiliation to a particular group by the method of infection of the hosts are presented in Table 3.
There was no apparent dominance of a particular rodent species. The highest occurrence was found for Myodes glareolus (45%) and Sylvaemus flavicollis (32%). These species dominated in the forest-steppe zone together with S. uralensis, although the last species occurs non-frequently in the samples (13%). The occurrence frequency of these species gradually decreased at the boundary zones between forests and completely deforested areas (Vlasov, 1996). Microtus arvalis occurs relatively rarely (8%) only at the open (woodless) areas in the site «Ostrasyevy Yary». Non-numerous records of Apodemus agrarius within the present study are in accordance with data of the last seven years that this species has been rarely recorded in the Belogorye State Nature Reserve. It is caused by unfavourable habitat conditions of ravine oak forests for this species (Vlasov, 1996;Shchekalo, 2017).  Step'» (Sury valley, Eremkin Log valley). Specificity of helminthes: oioxenous parasite -highly specific to a host species; stenoxenous parasite -specific at the level of host genus; oligoxenous parasite -specific at the level of a host family; euryxenous parasite -infests a broad range of non-related hosts.
The fodder base of micromammals included the food of both plant (seeds, vegetative parts of plants) and animal (Molluska, Myriapoda, Insecta) origin. Cricetidae species fed mainly on herbaceous plants. Muridae preferred cereal crops, but their food spectra also included terrestrial invertebrates (Terekhovich, 1966). The differences in the feeding types and the main food types could affect the invasion of rodents by certain helminth species.
The helminthes were divided into eight groups according to the way of penetration into the host body (Tokobaev, 1976). On the examined sites in the Belogorye State Nature Reserve, there were three groups of helminthes ( Table 3). First of them were invasive larvae in the eggs. Infection occurred when the eggs were swallowed (8 species). The second group was represented by invasive larvae living freely in terrestrial ecosystems. The infection occurred during ingestion of larvae (for example, together with plant leaves) (2 species). The third group includes invasive larvae localised in the body of terrestrial invertebrates (intermediate hosts). Infection of definitive hosts (small mammals) occurred, once the rodent consumed the intermediate hosts (9 species). We did not succeed in referring the representatives of the family Metastrongylidae to any group, because their biology has still not been studied sufficiently. Considering that the helminthes of the first, second (except Myodes glareolus), and the third group were found in all the studied hosts, we assumed that the diet of both Cricetidae and Muridae species included both plant and animal components and thus it was quite similar. LiI* Note: E -invasive larvae were in the eggs, infection occurred when the eggs were swallowed; FLL -invasive larvae live freely in terrestrial ecosystems, the infection occurred during ingestion of larvae; LiI -invasive larvae were localised in the body of terrestrial invertebrates (intermediate hosts), invasion of definitive hosts (small mammals) occurred when the rodent consumed the intermediate host; asterisk (*) indicates the helminthes that infect the rodents through prey (invertebrates) as a result of active hunting.
Single trematodes occurred only in the studied mouse-like rodents sampled on the site «Yamskaya Step'». Platynosomum muris Stcher-Stcherbakova, 1942 was found with low abundance (PP = 2.55%) only in four host specimens. Other species of the family Dicrocoeliidae (1.7%) were also recorded in low number. However, they have not been identified at species level. These helminthes were polyhostal. Their life cycle probably included terrestrial mollusks or insects as intermediate hosts, since there were no large water bodies on the sampling site.
Paranoplocephala omphalodes was found in Myodes glareolus, while Microtus arvalis was the main definitive host of this parasite in the neighbouring regions (Kursk region and Voronezh region) (Romashova & Romashov, 1996;Vlasov et al., 2015). We determined this species according to morphological characters. However, P. omphalodes is morphologically similar to some other species. In this case, it is preferable to analyse the nucleotide sequences, since there is no molecular genetic evidence that this parasite infects Myodes glareolus in the Central Russian Upland, nor in the whole of Russia (Vlasenko et al., 2019).
Taenia sp. (3.4%) was the dominant cestode species at the larval stages of development. PP of Hydatigera taeniaeformis (Batsch 1786) larvae and Mesocestoides sp. larvae were only 0.85%. As domestic cats (Felis catus Linnaeus, 1758) play a considerable role in the H. taeniae-taeniaeformis distribution, this parasite species can occur much more frequently on territories adjacent to Protected Areas.
According to the degree of specificity, euryxenous species prevailed among the registered cestodes (six species, including three at the larval ontogenetic stages). One species was oligoxenous (found in Cricetidae species). Finally, one species was stenoxenous (found only in Sylvaemus uralensis and Apodemus agrarius) ( Table 2).
According to the host specificity, the found nematodes were presented nearly equally by polyhostal (euryxenous) parasites (4 species) with a wide range of potential hosts and genusspecific (stenoxenous) parasites (4 species). One species, Syphacia petrusewiczi, was highly specific for Myodes glareolus ( Table 3).
Part of the specimens, designated as Syphacia sp. and similar to Sylvaemus flavicollis, was not identified to the species level, because the helminth specimens were damaged. These species were localised in the upper parts of the small intestine. It is not typical for most Syphacia species, while it is typical for Syphacia stroma. In addition, some morphological traits (e.g., the shape of the cephalic apex, distribution and number of papillae) indicated that these nematodes could belong to S. stroma. However, additional genetic analysis is necessary for confident species identification.
Long-term studies of the helminth fauna of mouse-like rodents have been also carried out in regions, adjacent to the Belgorod region (Kursk region (Vlasov et al., 2015;Vlasov, 2016), Voronezh region (Romashov & Shulyak, 1995;Ro-mashova & Romashov, 1996;Romashov, 1997;Romashov et al., 2003). However, these investigations concerned only Protected Areas. The comparison of these studies and our results demonstrates that in the Central Chernozem Region of Russia, the helminth fauna comprises in total over 60 species at the mature adult and larval ontogenetic stages of the five species of mouselike rodents. Amongst them there are Trematoda (at least eight and one species in mature adult and larval stages, respectively), Cestoda (at least 11 and nine species in mature adult and larval stages, respectively), Nematoda (at least 26 and two species in mature adult and larval stages, respectively), and Acanthocephala (only one species in the larval ontogenetic stage). Now, nine species have been recorded for all three mentioned regions, including Anoplocephaloides dentata (Galli-Valerio, 1905), Rausch, 1976, Paranoplocephala omphalodes, Spasskijela lobata, Hydatigera taeniaeformis s.l. (larvae), Trichuris muris, Heligmosomoides polygyrus, Syphacia nigeriana Baylis, 1928, S. petrusewiczi, and S. stroma. Four species (Plagiorchis elegans (Rudolphi, 1802), Catenotaenia matovi Genov, 1971, Heligmosomum costellatum (Dujardin, 1845, and Syphacia agrarian Sharpilo, 1973) were reported in Kursk region and Voronezh region, while they are still not registered in rodents of the Belgorod region. The first of those four species was recorded at the stage of cercaria in mollusks in the Belgorod region. Most likely, the remaining three species may also be found in the Belgorod region in future.
The larvae of trematodes Posterocirrus clethrionomi Andreiko & Khotenovsky, 1964, Skrjabinoplagiorchis vigisi Petrov & Merkuscheva, 1963, Psilotrema simillimum (Muhling, 1898), Notocotylus noyeri Joyeux, 1922, Echinostoma miyagawai Ishii, 1932, Echinostoma revolutum (Fröhlich, 1802, and Alaria alata (Goeze, 1782) were recorded only in the Voronezh region. This may be explained by the lack of large water bodies which is necessary to complete the life cycle of trematodes in the Kursk region and Belgorod region. The nematode Pterothominx sadovskoi is known only from the Belgorod region. In the present study, this species is noted for the first time in the Central Chernozem Region of Russia. Only one species of Acanthocephala, Macracanthorhynchus catulinus Kostylew, 1927 (larvae), was registered in mouse-like rodents in the Voronezh region. Its presence is associated with a specific intermediate host, the beetle Elenephorus sp.

Conclusions
In the present study, five species of mouselike rodents (Myodes glareolus, Microtus arvalis, Sylvaemus flavicollis, Sylvaemus uralensis, and A. agrarius) were registered in 2016-2017 at three clusters in the Belogorye State Nature Reserve («Les na Vorskle», «Ostrasyevy Yary», and «Yamskaya Step'»). Twenty helminth species were found. Of them, ten species belong to Nematoda, and ten species to Plathelminthes, including eight Cestoda species and two Trematoda species. In Cestoda, we found both mature adults (for which the rodents were definitive hosts) and larvae (for which the rodents were intermediate hosts). In the examined mammals, the parasite prevalence was 72%. We recorded parasites in 91% of the Muridae specimens and only in 55.6% of the Cricetidae specimens.
According to the host specificity, euryxenous species prevailed in flatworms. The nematodes were represented by polyhostal and host-specific species in almost equal proportions. Syphacia petrusewiczi, a highly specific parasite for Myodes glareolus, was also found.
Two species, Platynosomum muris (Trematoda, Dicrocoeliidae) and Pterothominx sadovs-sadovskoi (Nematoda, Capillariidae), were reported for the first time for the helminth fauna of the Central Chernozem Region of Russia.