The Diversity of Trematodes in Myotis Bats (Chiroptera, Vespertilionidae) from the Samarskaya Luka (European Russia)

Simple Summary Different species of bats often inhabit the same territories but have differences in diet. Bats become infected with parasitic worms such as trematodes when they feed on insects. Fluke diversity is determined by bats’ dietary breadth. The purpose of our research was to study trematode diversity in five Myotis species inhabiting Samarskaya Luka National Park. In the 2005–2007 period, we studied parasitic worms in bats and found 11 trematode species. One parasite species, Prosthodendrium cryptolecithum, was recorded for the first time in Russia. Only three fluke species were revealed in all studied bat species. Our study shows that trematode species diversity is higher in Myotis daubentonii and M. dasycneme. The trematode fauna of M. brandtii, M. nattereri, and M. mystacinus is less diverse. Our results confirm the similarity of habitats and diets among the five Myotis species and show weak competition among bats for food items. Abstract Various bat species often occupy the same habitats. Cohabitation should induce different preferences in spatial or trophic components of the bat ecological niche to reduce their competition. This determines the differences in the trematode fauna of Myotis spp. The purpose of our research was to study the biodiversity of trematodes in syntopic populations of five Myotis species in the Samarskaya Luka National Park. In the 2005–2007 period, we studied 867 bat specimens via the methods of complete helminthological dissection. In total, 11 trematode species from the families Plagiorchiidae (Plagiorchis koreanus, P. mordovii, P. muelleri, and P. vespertilionis), Pleurogenidae (Parabascus duboisi), and Lecithodendriidae (Prosthodendrium ascidia, Pr. chilostomum, Pr. cryptolecithum, Pr. hurkovaae, Pr. longiforme, and Lecithodendrium linstowi) were found in five Myotis species. Only three trematode species, P. koreanus, Pr. chilostomum, and P. duboisi, are common to all studied Myotis spp. Prosthodendrium cryptolecithum was recorded for the first time in Russia. Trematode species diversity is higher in Myotis daubentonii and M. dasycneme. The trematode fauna of M. brandtii, M. nattereri, and M. mystacinus is less diverse. The determining factor in the infection of bats with trematodes is feeding on semi-aquatic insects, possible second intermediate hosts of the parasites. The infection of bats with flukes occurs at different levels of host (Myotis spp.) abundance as a result of the realization of the main bat trophic relationships. Our results confirm the data that the ecological niches of the five Myotis species partially overlap. Analysis of the trematode fauna in Myotis spp. showed that, in the Samarskaya Luka, there may be weak competition for food items among bats.


Introduction
Recently, more and more attention has been paid to the study of groups of co-inhabiting and even taxonomically close species of bats.In such communities, rather complex and contrasting relationships develop between their members [1][2][3][4][5].Various bat species often occupy the same habitats.In this case, the principle of competitive exclusion can be applied, Karelia [56].Gulyaev and co-authors [57] provided data on one studied specimen of M. brandtii from the Magadan Oblast, in which only Plagiorchis sp. was found.Previously, we studied the helminths in M. dasycneme and M. daubentonii from Mordovia, where three and six trematode species were identified in bats, respectively [58,59].Our previous works, devoted to parasites of vertebrates from the Middle Volga region, include information about flukes in all five chiropteran species [60][61][62][63][64].
Thus, the aim of our present research was to study the biodiversity of trematodes in syntopic populations of five Myotis species from the Samarskaya Luka (European Russia).

Materials and Methods
The material for this research was collected during our own field studies on bat ecology and helminths in the territory of the Samarskaya Luka National Park (Samara Oblast, European Russia), which were conducted in the 2005-2007 period.The study area was the coastal zone of the Volga River in the northern part of the Samarskaya Luka (Samara Oblast).Bats were studied at three trapping stations of the National Park near Solnechnaya Polyana, Bogatyr and Shiryaevo villages (Figure 1).
Animals 2023, 13, x FOR PEER REVIEW 3 of 13 nezh Nature Reserve.Demidova and Vekhnik [55] studied trematodes in M. mystacinus and M. brandtii from the Samarskaya Luka, in which two and nine trematode species were identified, respectively.Five species of trematodes were found in M. brandtii from Karelia [56].Gulyaev and co-authors [57] provided data on one studied specimen of M. brandtii from the Magadan Oblast, in which only Plagiorchis sp. was found.Previously, we studied the helminths in M. dasycneme and M. daubentonii from Mordovia, where three and six trematode species were identified in bats, respectively [58,59].Our previous works, devoted to parasites of vertebrates from the Middle Volga region, include information about flukes in all five chiropteran species [60][61][62][63][64]. Thus, the aim of our present research was to study the biodiversity of trematodes in syntopic populations of five Myotis species from the Samarskaya Luka (European Russia).

Materials and Methods
The material for this research was collected during our own field studies on bat ecology and helminths in the territory of the Samarskaya Luka National Park (Samara Oblast, European Russia), which were conducted in the 2005-2007 period.The study area was the coastal zone of the Volga River in the northern part of the Samarskaya Luka (Samara Oblast).Bats were studied at three trapping stations of the National Park near Solnechnaya Polyana, Bogatyr and Shiryaevo villages (Figure 1).In total, we studied 247 M. brandtii, 262 M. daubentonii, 135 M. dasycneme, 125 M. mystacinus, and 98 M. nattereri.Bats were caught with mist nets at night.We used the common method of placing the net between two vertical sticks, which were 6 m long telescopic fishing rods [65].We also studied bats that died of natural causes in their wintering sites.In total, we studied 247 M. brandtii, 262 M. daubentonii, 135 M. dasycneme, 125 M. mystacinus, and 98 M. nattereri.Bats were caught with mist nets at night.We used the common method of placing the net between two vertical sticks, which were 6 m long telescopic fishing rods [65].We also studied bats that died of natural causes in their wintering sites.
To characterize the helminth infection of Myotis bats, the following indices, which are generally accepted in parasitology, were used: prevalence (P, %), mean abundance (MA), intensity range (I, specimens), and mean intensity (MI) [69,70].To determine the species diversity of trematodes, Shannon (H') and Shannon evenness (E) indices were calculated.The significance of differences between Shannon index values was measured using Student's t-test.The degree of similarity between the trematode faunas of Myotis spp. was revealed using the Jaccard (C J ) (qualitative data) and Sørensen (C N ) (quantitative data) similarity indices [71].The degree of similarity was assessed as low (0-0.33),medium (0.34-0.66), and high (0.67-1).
The dominance of species in the parasite community was determined using the Palia-Kovnatsky dominance index (D) [72].Trematode dominance groups were as follows: 10-100, dominants; 1-10, subdominants; 1-0.001, adominants.The Mann-Whitney (U) test was used to compare the total infections of Myotis spp.and assess the significance of differences in infection with individual trematode species.A dendrogram of similarity of helminth faunas of Myotis spp. was obtained using the unweighted pair group method with arithmetic average (UPGMA) and the Morisita index as a distance measure in PAST 2.17 [73].To standardize bat sample size, we used the species richness index (bootstrap estimator), which predicts the number of helminth species not included in collections [74,75].The taxonomy of flukes was provided according to Fauna Europaea (http://www.fauna-eu.org/, accessed on 3 August 2023) [76]

Results
A total of 11 species of trematodes were revealed in five bat species from the Samarskaya Luka National Park (Table 1).
In Myotis mystacinus, as well as in M. nattereri, six species of trematodes were found.The total infection of M. mystacinus with flukes was 60%, MA = 12.8.Among trematodes identified in M. mystacinus, Pr. ascidia is dominant (59.3), while P. koreanus (5.6) is subdominant.The total infection of M. nattereri compared to other Myotis bats is low and amounted to only 32%, and the MA was 2.6.In the trematode fauna of M. nattereri, the dominant is P. koreanus (27.1), the subdominant is Pr.ascidia (1.7).The remaining four species in the trematode fauna of M. nattereri are adominants.
In the trematode fauna of Myotis bats, the structure and number of dominant and subdominant species are different (Figure 2).For all five Myotis species, no common dominant or subdominant trematode species was identified.Only two species of trematodes, P. koreanus and Pr.ascidia, are the dominant or subdominant for four bat species, and P. duboisi for three Myotis species (Figure 2).
Only three of the eleven trematode species were found together in all five bat species: P. koreanus, Pr. chilostomum, and P. duboisi (Table 1).Prosthodendrium ascidia was found in four species of bats.Plagiorchis vespertilionis, P. mordovii, P. muelleri, Pr. hurkovaae, and L. linstowi were each recorded in three host species.Two host species were noted for Pr.cryptolecithum and Pr.longiforme (Table 1).
Values of trematode species diversity in Myotis spp.from the Samarskaya Luka National Park are shown in Table 2.The diversity indices of the trematode fauna are significantly higher for M. daubentonii and M. dasycneme, with the exception of the Margalef index values.The values of the Margalef index for these bat species are slightly lower than for M. brandtii and M. nattereri (Table 2).The trematode fauna of M. brandtii M. nattereri are less diverse.The least diversity in the trematode fauna was observed in M. mystacinus (Table 2).This bat species has a significantly lower Shannon diversity, Shannon evenness, Margalef and Simpson evenness indices than other species (Table 2).The differences in the Shannon species diversity index among the trematode fauna of five chiropteran species are significant (p < 0.001), with the exception of the pair M. daubentonii-M.dasycneme (p > 0.05).
A comparison of trematode infection of five Myotis species using the Kruskal-Wallis test revealed significant differences (H = 425.6,p < 0.0001).A pairwise comparison of infection of different Myotis spp.using the Mann-Whitney test revealed significant differences in most cases (p < 0.0001), with the exception of the pair M. daubentonii-M.dasycneme (Table 3).An analysis of the infections of bats with common trematode species using the Mann-Whitney test showed significant differences in infection with Pr. chilostomum (in all cases), P. koreanus, and P. duboisi (in most cases) (Table 3).Differences in the infection with P. koreanus in pairs M. brandtii-M.dasycneme, M. mystacinus-M.nattereri, M. nattereri-M.daunentonii, as well as in the infection of M. mystacinus and M. nattereri with P. duboisi, were not significant.There were no significant differences in the infection with P. koreanus in pairs M. brandtii-M.dasycneme, M. mystacinus-M.nattereri, M. nattereri-M.daunentonii, as well as in the infection of M. mystacinus and M. nattereri with P. duboisi (Table 3).
We conducted a comparative analysis of the helminth faunas of Myotis species inhabiting the Samarskaya Luka.A similarity dendrogram of the parasite fauna of five bat species is presented in Figure 3.
P. duboisi, were not significant.There were no significant differences in the infection with P. koreanus in pairs M. brandtii-M.dasycneme, M. mystacinus-M.nattereri, M. nattereri-M.daunentonii, as well as in the infection of M. mystacinus and M. nattereri with P. duboisi (Table 3).
We conducted a comparative analysis of the helminth faunas of Myotis species inhabiting the Samarskaya Luka.A similarity dendrogram of the parasite fauna of five bat species is presented in Figure 3.A cophenetic correlation coefficient of 0.802 confirms the validity of the cluster.As a result of the cluster analysis, the considered Myotis species were divided into two clusters with the most similar helminth fauna (Figure 3).
Flukes parasitizing M. mystacinus and M. nattereri form the first cluster, in which the greatest similarity of trematode communities is observed (0.83).These species of bats differ maximally in trematode composition from the three other Myotis spp.thatare included in the second cluster (Figure 3).
The second cluster is formed by a clade of trematode faunas of M. brandtii and M. daubentonii (0.78) that is close to the trematode fauna of M. dasycneme, more similar to that of M. brandtii (0.78), and less similar to that of M. daubentonii (0.75) (Figure 3).The remaining pairs showed an average degree of similarity ranging from 0.57 to 0.63.

Discussion
Analysis of the trematode fauna of Myotis spp.from the Samarskaya Luka National Park showed that the species composition was the richest in M. brandtii (10 species).Slightly fewer trematode species were found in M. daubentonii and M. dasycneme (eight species each), and the smallest number of trematode species was found in M. mystacinus and M. nattereri-six species each (Table 1).A cophenetic correlation coefficient of 0.802 confirms the validity of the cluster.As a result of the cluster analysis, the considered Myotis species were divided into two clusters with the most similar helminth fauna (Figure 3).
Flukes parasitizing M. mystacinus and M. nattereri form the first cluster, in which the greatest similarity of trematode communities is observed (0.83).These species of bats differ maximally in trematode composition from the three other Myotis spp.thatare included in the second cluster (Figure 3).
The second cluster is formed by a clade of trematode faunas of M. brandtii and M. daubentonii (0.78) that is close to the trematode fauna of M. dasycneme, more similar to that of M. brandtii (0.78), and less similar to that of M. daubentonii (0.75) (Figure 3).The remaining pairs showed an average degree of similarity ranging from 0.57 to 0.63.

Discussion
Analysis of the trematode fauna of Myotis spp.from the Samarskaya Luka National Park showed that the species composition was the richest in M. brandtii (10 species).Slightly fewer trematode species were found in M. daubentonii and M. dasycneme (eight species each), and the smallest number of trematode species was found in M. mystacinus and M. nattereri-six species each (Table 1).
Analysis of the species diversity of trematodes in Myotis spp.showed that the trematode fauna are more diverse for M. daubentonii and M. dasycneme, despite the fact that a larger number of trematode species were found in M. brandtii (Tables 1 and 2).The value of the Shannon diversity index for M. brandtii is lower, which is associated with the high abundance and dominance of one species in its trematode fauna, Pr. ascidia (Figure 2).At the same time, the Margalef index, which takes into account species richness and the total number of parasite specimens, is significantly higher in M. brandtii, from which the largest number of trematodes was collected (Table 2).The abundance and dominance of Pr. ascidia in M. mystacinus also leads to a significant decrease in the Shannon index.As a result, this species of bats has the lowest species diversity of the studied flukes (Table 2, Figure 2).
All trematode species were found in Myotis spp. at the mature stage and are hostspecific parasites of bats.Three of the eleven recorded trematode species (Pr.ascidia, Pr. cryptholecithum, and P. duboisi) in bats in the study area were found only in Myotis spp.In addition to Myotis spp., other species of bats from the Samarskaya Luka also participate in the life cycles of eight more species of trematodes [60,61,63].
Infection of bats with trematodes occurs entirely when feeding on semi-aquatic insects, which serve as second intermediate hosts of these parasites.Thus, findings of P. koreanus and P. vespertilionis indicate the consumption of adult forms of Diptera, Ephemeroptera, Megaloptera, Trichoptera, and Odonata by bats [31].The presence of Pr. chilostomum in the helminth fauna of Myotis spp.indicates that bats consume adults of Trichoptera and, possibly, Odonata [31].The second intermediate hosts of Pr. ascidia are the chironomid larvae, such as Chironomis plumosus (Linnaeus, 1758) [31,77].
The life cycles of P. mordovii, P. muelleri, L. linstowi, Pr. longiforme, Pr. cryptolecithum, Pr. hurkovaae, P. duboisi, and P. lepidotus are currently not studied.Probably, the second intermediate hosts of these trematodes, like other bat trematodes, are insects that develop in the aquatic environment [31].
Trematodes are parasites with a complex life cycle, including a change of hosts.Accordingly, the trematode fauna of Myotis spp.depends on the presence/absence of various first (freshwater gastropods) and second (semi-aquatic insects) intermediate hosts at each site.In turn, the species composition of trematodes and their infection of intermediate hosts are determined by the diversity and abundance of final hosts (bats), which ensure the release of trematode eggs into the external environment.
According to Smirnov and Vekhnik [12], among Myotis spp., in terms of relative abundance index, M. daubentonii (10.3%) is the most numerous in the Samarskaya Luka; M. dasycneme (7.5%) and M. brandtii (6.5%) are slightly less common.M. mystacinus (2.3%) and M. nattereri (0.2%) are rare in the study area.Analysis of the trematode fauna of bats showed that the most abundant species, M. daubentonii, does not have the largest number of trematode species (eight) but the same number as in M. dasycneme (Table 1).Myotis brandtii, inferior in relative abundance to M. daubentonii and M. dasycneme, nevertheless has the largest number of trematode species (10).The least abundant species, M. nattereri, has the same number of trematodes as M. mystacinus (six species each) (Table 1).When analyzing the infection of bats with trematodes, significant differences were established, which confirm that the highest parasitic load of trematodes is carried by M. daubentonii and M. dasycneme, to a lesser extent than M. brandtii, and the lowest parasitic load on Myotis spp. is revealed in M. mystacinus and M. nattereri (Tables 1 and 3).
It should be noted that the infection of bats with flukes occurs at different levels of host abundance (Myotis spp.) as a result of the realization of the main trophic relationships between chiropterans.The presented differences in the trematode fauna of various Myotis species are mainly associated with preferences for certain food items.The widest feeding range was found in M. daubentonii, whose diet in the Samarskaya Luka includes 10 food items from nine orders of insects.The diet of M. dasycneme, M. brandtii, and M. mystacinus is slightly smaller and contains nine food objects from eight orders of insects [12].Myotis nattereri also has nine food items, but from seven orders of insects [12].
Despite the similar composition of the diet, the proportion of various food items of the five bat species is not the same [12].Thus, the main diet of M. daubentonii and M. dasycneme consists of Trichoptera, Lepidoptera, and Diptera.Coleoptera are somewhat less represented in the diet of these two species, and among the food items for M. daubentonii there are also representatives of the orders Hemiptera and Homoptera.Insects of other orders are rarely found in the diet of M. daubentonii and M. dasycneme [12].
The diet of M. brandtii is dominated by Lepidoptera and Diptera; Coleoptera are rather less represented; insects of the orders Trichoptera, Neuroptera, Homoptera, and Hymenoptera are rarely found [12].In M. mystacinus, the main diet components are Lepidoptera; Trichoptera are less represented, and Coleoptera, Homoptera, and Diptera are rare.The diet of M. nattereri is dominated by Lepidoptera.Members of Trichoptera, Coleoptera, Diptera, Hymenoptera, and Homoptera are present in the diet in equal proportions [12].
The identified differences in the infection of bats with common trematode species can be explained by the different proportions of food items (second intermediate hosts of trematodes) in the diet of Myotis spp.
The diversity of the trematode fauna of bats also depends on the number of hunting grounds that bats visit.Visiting more hunting sites expands the bats' diet, which ultimately increases the likelihood of their infection with various species of trematodes.
Myotis spp.show unequal preferences for different types of hunting grounds.Among bats, the largest number of hunting sites was found for M. brandtii, M. daubentonii, and M. dasycneme [78].Myotis nattereri prefers to hunt in only one type of hunting sites, whereas M. mystacinus has two types of hunting grounds [78].Our identification of a greater number of trematode species in M. brandtii, M. daubentonii, and M. dasycneme, compared to that in M. nattereri and M. mystacinus, confirms these data on hunting preferences (Table 1).
In addition, Myotis spp.occupy different spatial niches when hunting sites overlap [78].Thus, M. daubentonii and M. dasycneme hunt above the water surface, M. brandtii feeds high in the tree crowns, and M. mystacinus hunts immediately below (the middle and lower parts of the tree crowns).Myotis nattereri prefers to hunt in open confined spaces above the ground, such as forest edges, wide forest glades, and paths [78].The association of different Myotis species with certain hunting grounds also affects the diversity and abundance of insects in the diet of bats.Ultimately, this determines the species composition and abundance of trematodes in Myotis spp.
A comparative analysis of the trematode fauna of five Myotis species inhabiting the Samarskaya Luka National Park showed a significant similarity in the composition of trematodes in different bat species (Figure 3).This is due to their similar lifestyle and feeding on semi-aquatic insects, which serve as second intermediate hosts of flukes.Thus, the finding of three common trematode species in bats (P.koreanus, Pr. chilostomum, and P. duboisi) indicates that they feed on the same food items (insects)-the second intermediate hosts of these trematode species.Our data confirm the partial overlap of spatial and trophic niches of various Myotis spp.and their division according to food preferences and hunting grounds.

Conclusions
Eleven trematode species were found in five species of Myotis spp.from the Samarskaya Luka National Park.The trematode Pr. cryptolecithum was recorded in bats of Russian fauna for the first time.
Analysis of the trematode species diversity in Myotis spp.showed that the trematode fauna are more diverse in M. daubentonii and M. dasycneme (8 species each), despite the fact that a larger number of trematode species were found in M. brandtii (10).The lowest species diversity of trematodes was observed in M. mystacinus, while the number of trematode species noted in M. mystacinus and M. nattereri is the same (six species each).All Myotis species participate to varying degrees in the realization of the life cycles of trematodes on the territory of the Samarskaya Luka.However, abundant bat species (M.daubentonii, M. dasycneme, and M. brandtii) carry the main parasitic load and have both the highest trematode species diversity and high infection rates.Despite the relatively low abundance of M. mystacinus and M. nattereri and their low infection with trematodes, these species are also involved in the distribution and preservation of the abundance of the parasites.
The determining factor in the infection of bats with trematodes is feeding on semiaquatic insects (second intermediate hosts).Therefore, the infection of bats with trematodes occurs at different levels of host abundance (Myotis spp.) as a result of the realization of the main trophic relationships.
The diversity of the trematode fauna of Myotis spp. is associated with the number of hunting sites that bats visit.Various Myotis species visiting the same hunting grounds and consuming the same food items (but in different proportions) have an average degree of similarity in the trematode fauna.The revealed differences in the trematode fauna of Myotis spp.are associated both with the food preferences of various species and with the variety of hunting grounds they use.
Our parasitological results confirm the data on the partial overlap of the ecological niches of the five Myotis species in spatial and trophic components.An analysis of the trematode fauna of Myotis spp.showed that, in the territory of the Samarskaya Luka, there may be weak competition for food items among bats due to their specialization and divergent habitats.

Figure 1 .
Figure 1.Map of bat trapping sites in the Samarskaya Luka National Park.Red crosses on the map indicate the trapping sites.

Figure 1 .
Figure 1.Map of bat trapping sites in the Samarskaya Luka National Park.Red crosses on the map indicate the trapping sites.

Figure 2 .
Figure 2. Dominant and subdominant species of trematodes in Myotis bats from the Samarskaya Luka (D -the Palia-Kovnatsky dominance index).

Figure 2 .
Figure 2. Dominant and subdominant species of trematodes in Myotis bats from the Samarskaya Luka (D -the Palia-Kovnatsky dominance index).
. Trematode voucher specimens qwee stored in the parasitological collection of the Institute of Ecology of the Volga River Basin of the Russian Academy of Sciences (Togliatti, Russia).

Table 1 .
Trematodes in Myotis spp.from the Samarskaya Luka National Park.
Note:1-prevalence of infection (P), in brackets-intensity range (I), after brackets-mean intensity (MI), beyond the line-mean abundance (MA); 2 -in brackets-predicted number of species not included in collections due to insufficient host sampling.

Table 2 .
Values of trematode biodiversity indices in Myotis spp.

Table 3 .
Validity of differences in the infection of Myotis spp. with common trematode species.