Helminth Community Structure of Tits Cyanistes caeruleus and Parus major (Paridae) during Their Autumn Migration on the Southern Baltic Coast

Simple Summary Parasites of birds can be an indicator of environmental quality, but this issue is very poorly understood. Migratory birds can transfer parasites between ecosystems, thus increasing the size of their populations. The biodiversity of bird parasites and the predominance of specific parasite species can be influenced, as in the case of non-parasitic animals, by global changes in the environment, including climate change and anthropogenic transformation of ecosystems. The aim of the present research was to describe and compare helminth communities of Eurasian blue tit Cyanistes caeruleus and the Great tit Parus major, at the component community and infracommunity level, on the southern coast of the Baltic Sea during autumn migration to their wintering grounds. The main findings of the research were the presence of several parasite species that had not previously been recorded in these tits in Poland, the dominance of the digenetic fluke, and the high prevalence of the filarial nematodes. Abstract The research problem undertaken in this study is to determine the scale of infection of Eurasian blue tit Cyanistes caeruleus and Great tit Parus major and the biological diversity of their internal parasites, helminths. The aim of the study is to gain new knowledge about the structure of the helminth communities of the Eurasian blue tit and Great tit on the southern coast of the Baltic Sea during autumn migration to their wintering grounds. Helminths of tits were collected in 2008–2012 on the southern coast of the Baltic Sea in Poland. PAST v. 2.11 software was used for the calculations. Barcoding DNA was used to identify trematodes initially classified based on morphological characters to the genera Leucochloridium and Urogonimus. Cestodes Anonchotaenia globata were recorded for the first time in Poland. The Eurasian blue tit is a new host in Poland for three species of helminths: cestode Monosertum parinum and filarial nematodes, Cardiofilaria pavlovskyi, and Diplotriaena henryi. The Great tit is a new host in Poland for trematode Urogonimus macrostomus, cestode A. globata and M. parinum, and filarial nematode Diplotriaena obtusa. The nematode C. pavlovskyi was the species most frequently recorded in both host species. A high degree of similarity was found between the component communities and infracommunities of helminths in Eurasian blue tit and Great tit. The new information provided in this study has increased our knowledge of the transmission of helminths in Central Europe.

similar food preferences, which can include the intermediate hosts of parasites. However, there are reasons to believe that the structure of helminths may differ in the two species; for example, they prefer different biotope types and feed in different ways. The biotopes inhabited by the Great tit include poor environments, such as coniferous forests and urban areas, while the Eurasian blue tit chooses biotopes more closely associated with deciduous forests and parks. The Great tit readily feeds on the ground, while the Eurasian blue tit avoids feeding on the ground, preferring to penetrate tree crowns and shrubs.

Sampling
The birds were collected during the autumn migration season, from September to November 2008-2012. A total of 67 Eurasian blue tit individuals and 166 Great tit individuals were subjected to parasitological analysis. Age was established in 30 Eurasian blue tits (16 juveniles and 14 adults) and 95 Great tits (73 juveniles and 22 adults). Sex was determined in 30 Eurasian blue tits (17 females and 13 males) and 101 Great tits (58 females and 43 males), although in case of some individuals, both age and sex, or only one of the features, were determined. The birds inhabited northern Poland and were collected along the southern coast of the Baltic Sea. Most birds were obtained during sampling organised by Operation Baltic. No birds were sacrificed for the purpose of sampling. The migration process may deteriorate body condition and increase the mortality rate of birds due to numerous and carious factors [13][14][15]. Birds used for the research had died of various causes, e.g., due to a sudden and extremely unfavourable change in weather conditions on the Baltic coast, attack by predators, or collision with an obstacle, such as a window. The birds for parasitological examination were not classified according to the cause of death, and their body condition was not evaluated. Dead birds were not always fresh, as all available birds were used for testing in accordance with the literature recommendations [14,[16][17][18], but the internal organs of all birds had adequate conditions for collecting parasites. The freshness of the birds was primarily a factor for cestodes, which are delicate and highly susceptible to lysis, while decomposition had a relatively minor effect on trematodes and nematodes, specimens of which were in very good condition for species identification. For this reason, not all cestode specimens were precisely identified by genus and species. Birds in which cestode species could not be identified or for which other data were unavailable were excluded from the analysis of ecological indices describing the structure of the component community and infracommunity of helminths. All procedures were in compliance with Polish law. Consent for the collection of dead birds and parasitological research was obtained from the General Director for Environmental Protection and the Regional Director for Environmental Protection in Szczecin.

Sample Processing and Parasite Identification
Parasites were preserved in 70-75% ethanol. Trematodes were stained with alum carmine and cleared in clove oil, and microscope slides were mounted with Canada balsam. Cestodes were stained with acetocarmine. Scolices were cleared in Faure's medium. Nematodes were cleared in glycerol. Morphological examination and identification of parasites were carried out using keys and original works [19][20][21][22][23][24][25][26]. DNA barcoding was used to identify 31 trematode specimens initially classified on the basis of morphological characters to the genera Leucochloridium and Urogonimus (Leucochloridiidae); further, identification was not possible due to the high similarity or variability of the main diagnostic features within the genera (a wide range of body shape variation, the size ratio of suckers, overlapping of internal organs, or their concealment behind uterine loops filled with dark eggs in adult trematodes). For barcoding, genomic DNA was isolated from each individual using the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany). Next, a fragment (~300 bp) of cytochrome c oxidase subunit I (COI) was amplified using primers JB3 and JB4.5 [27] and JB3 and JB4.5-in [28]. The composition of 20 µL of the reaction mixture was as follows: 5x Phire Reaction Buffer with Mg 2+ , 0.2 mM dNTP, 0.5 µM of each primer, 0.4 µL Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA), and 6.0 µL template DNA. PCR reactions were carried out in a T100TM Thermal Cycler (Bio-Rad, Hercules, California, USA) in 40 cycles, according to the following temperature profile: initial denaturation at 98 • C for 5 min, then 98 • C for 5 s, 50 • C for 5 s, 72 • C for 20 s, with final elongation at 72 • C for 1 min. Individual PCR products were identified on a 1.5% agarose gel in the presence of the Nova 100 bp DNA Ladder mass standard (Novazym, Poznań, Poland). DNA sequencing of all PCR products was performed by Genomed, Poland, and the raw reads were assembled with Geneious 8.0 [29] and compared against the GenBank sequences using BLAST [30]. Species identity of the flukes from this study was confirmed based on the alignments with COI homologous sequences retrieved from the GenBank database using BLAST search [30]. Then, the genetic diversity of the flukes (number of haplotypes, haplotype diversity, and nucleotide diversity) was calculated using DNAsp 5.10 software [31]. The ML tree was constructed using MEGA5 based on the sequences of haplotypes of Parus major and Cyanistes caeruleus flukes identified in this paper and sequences of other flukes retrieved from the GenBank [32]. The Ornithobilharzia canaliculata (GenBank acc. no. AY157194.1) COI sequence was used as the outgroup.

Data Analysis
Basic parasitological parameters were calculated: prevalence, as the percentage of infected hosts among all tested birds; mean intensity, i.e., the number of parasites divided by the number of infected hosts; range of intensity, i.e., the minimum and maximum number of parasites; and mean abundance, i.e., the number of parasites divided by the number of all hosts, infected and not infected. All parameters were defined according to Bush et al. [33]. The component community structure was analysed, specifying the community of all parasites in the Eurasian blue tit population and all parasites in the Great tit population, as well as the infracommunity structure, meaning the community of parasites in one host individual. For each component community, biological diversity was determined using Simpson's index and dominance using the Berger-Parker index. The Brillouin index was used for determination of infracommunity diversity. All indices are defined according to Magurran [34]. PAST v. 2.11 software [35] was used for the calculations. Parasitological parameters and ecological indices of the Eurasian blue tit and Great tit were compared. Parasitological parameters were also compared by sex and age of birds. Prevalence was compared using the chi 2 test. The nonparametric Mann-Whitney U test was used to test differences in the intensity, abundance, richness, and Brillouin index between communities of the Eurasian blue tit and Great tit. Statistical tests were carried out in Statistica 13.1 software.

Faunistic and Molecular Analyses
New faunistic data were obtained in this research. Cestodes Anonchotaenia globata (von Linstow, 1879) were recorded for the first time in Poland  The molecular analyses conclusively identified the trematodes with atypical morphological characters to two species: Leucochloridium paradoxum (Carus, 1835) (n = 21) and U. macrostomus (n = 10) ( Figure 1). Their taxonomic identity was confirmed in GenBank at the level of identity of alignments to homologous sequences above 98%. Maximum likelihood estimation also showed that in P. major individuals (nos. 4 and 7), there was only one species of fluke (letter designations a, b, etc.). The assessment of genetic diversity showed a greater number of haplotypes (n = 6) and lower levels of haplotype diversity (0.48 ± 0.13) and nucleotide diversity (0.0026) in trematodes L. paradoxum than in specimens of U. macrostomus (n = 4, 0.64 ± 0.15 and 0.0144, respectively). Further analysis showed that all haplotypes of U. macrostomus (PL-US-HU1-PL-US-HU4) and five of the six haplotypes of L. paradoxum (PL-US-HL1-PL-US-HL5) had not previously been described, so they were deposited in the GenBank database (MK882500-MK882508).

Structure of the Component Communities and Infracommunities of Helminths
Among parasite species, only the prevalence of L. paradoxum showed statistically significant differences between component communities of the tit species (p = 0.027) ( Table 1). The prevalence of helminths was higher in the Great tit than in the Eurasian blue tit (62.7% vs. 44.8%, p = 0.012) ( Table 2).

Structure of the Component Communities and Infracommunities of Helminths
Among parasite species, only the prevalence of L. paradoxum showed statistically significant differences between component communities of the tit species (p = 0.027) ( Table  1). The prevalence of helminths was higher in the Great tit than in the Eurasian blue tit (62.7% vs. 44.8%, p = 0.012) ( Table 2).  n-number of infected birds, T-Trematoda; C-Cestoda; N-Nematoda; *-statistically significant difference between parameter of infection of Eurasian blue tit and Great tit, p ≤ 0.05.
The mean intensity and mean abundance of helminths were higher in the Great tit than in the Eurasian blue tit, but only the difference in mean abundance was statistically significant (11.4 vs. 6.0, p = 0.024). There were five helminth taxa parasitizing the Eurasian blue tit and eight in the Great tit ( Table 2). The component community and infracommunity of helminths of the Eurasian blue tit and Great tit did not differ; Simpson's index, the Berger-Parker index, and Brillouin index were similar in the two species of birds and indicated strong dominance of L. paradoxum (Table 2).

Helminth Infection According to Host Age and Sex
In the juveniles of Eurasian blue tit, trematodes and nematodes were identified, while in the adult birds, additional parasitic cestodes were found. The differences between parasitological parameters in juvenile and adult Eurasian blue tits were not statistically significant (Table 3). Table 3. Occurrence of helminths in juvenile and adult tits and in female and male tits. Numbers of examined juveniles and adults and females and males are given in brackets.   Trematodes, cestodes, and nematodes were found in juvenile and adult Great tits, but the prevalence of cestodes was much higher in adult Great tits (31.8%) than in juveniles (2.7%), and the difference was statistically significant (p = 0.000) (Table 3). Trematodes, nematodes, and cestodes were found in female Eurasian blue tits, but males were infected only by nematodes. The differences between parameters of infection of male and female Eurasian blue tits were not statistically significant. Trematodes, cestodes, and nematodes were present in both female and male Great tits, and the differences between parameters of infection in females and males of Great tits were not statistically significant (Table 3).

Discussion
The presence of parasites as factors regulating the size of host populations can weaken the host organism. Clinical symptoms and the mortality of birds caused by parasites are noted in the literature [36,37]. Although it is widely believed that in the natural environment, parasites do not cause severe pathological changes in wild animals, in contrast to livestock and humans, it seems unlikely that several hundred or even a few dozen trematodes of the genus Leucochloridium in a bird's gut, Cardiofolaria nematodes in the pericardium and coelom, or Diplotriaena in the pulmonary cavity would not disturb the function of these organs or affect the condition of seasonally migrating birds. For example, large numbers of flukes L. paradoxum in the cloaca of birds have been reported in the Czech Republic, with the suggestion that this may lead to emaciation and the consequent death of the birds [36].

Similarities and Differences in Infection of Eurasian Blue Tits and Great Tits: Different Parasite Composition but Similar Helminth Community Structure
The present results showed that during the migration of Eurasian blue tit and Great tit over the southern Polish coast of the Baltic Sea, the helminths of the two species have a different species composition but similar community structure. The study revealed seven parasite-host relationships previously unknown in Poland: between Eurasian blue tit and nematodes (C. pavlovskyi and D. henryi) and cestodes (M. parinum), and between Great tit and nematodes (D. obtusa) trematodes (U. macrostomus) and cestodes (M. parinum and A. globata). These parasites had previously been recorded in Poland in other bird species [38]. Among the nine identified helminth species, four were common to both tit species: L. paradoxum, M. parinum, C. pavlovskyi, and D. henryi. The dominant species in the communities of the tits in terms of prevalence was C. pavlovskyi, while L. paradoxum, whose prevalence can be described as intermediate, was clearly dominant in terms of the abundance of individuals in the helminth communities. The values for the parasitological parameters of M. parinum and D. henryi were low, like those of other helminths found only in the Eurasian blue tit (Plagiorchis maculosus (Rudolphi, 1802)) or only in the Great tit (U. macrostomus, A. globata, Capillaria triedens (Dujardin, 1845) and D. obtusa), which should be regarded as accessory species.
The presence of L. paradoxum and U. macrostomus among trematode specimens that could not be assigned to species on the basis of morphological characters was confirmed by molecular analysis. Trematodes L. paradoxum and U. macrostomus are typical species found in tits in Poland and other European countries. The DNA barcoding method used for genetic identification, based on analysis of variation in the COI gene sequence, proved successful in resolving doubts regarding the identification of trematodes found in Eurasian blue tit and Great tit as L. actitis and L. macrostomum. The results of the molecular analysis clearly confirmed the presence of two species of trematodes: L. paradoxum and U. macrostomus. L. paradoxum was shown to have more haplotypes and lower haplotype and nucleotide diversity than U. macrostomus. The differences were most likely due to the fact that the number (n = 21) of L. paradoxum trematodes identified was higher than the number of trematodes of the species U. macrostomus (n = 10). The genetic analyses included in this study were mainly meant to identify the species of trematodes that could not be classified on the basis of morphological characteristics. Therefore, confirmation and verification of the genetic diversity among the collected representatives of L. paradoxum and U. macrostomus require more extensive analyses based on a larger group of representatives of both species.
Following the views of Freeland [39] regarding factors determining the distribution of parasitic species in potential host species, i.e., phylogenetic origin, size, morphology, and nutritional requirements, several possible reasons for the similarities and differences in the infection of the Eurasian blue tit and Great tit can be postulated. The similarities, i.e., the high prevalence of C. pavlovskyi, the lack of significant difference in the intensity of helminth infection in the two tit species, the low Simpson's index, the high Berger-Parker index indicating the dominance of L. paradoxum in the communities of the two host species, the lack of significant difference between the diversity of the helminth infracommunity of the Eurasian blue tit and Great tit, and the lack of significant difference between the number of helminth species in the helminth infracommunity of the two tit species, may have several explanations: --Both species of birds prefer similar biotopes, such as forests, parks, and gardens, and they can both be found in the same areas. Filarial nematode C. pavlovskyi is a generalist parasite, found mainly in passerines but also in other birds [21,[40][41][42]. Its life cycle is indirect and includes a dipteran of the genus Armigera, Culex, or Mansonia [43]. Scientists currently consider the changing ranges of mosquitoes (Culicidae) as one of the effects of climate change. This interrelation is explored more extensively in the study of mosquito-borne human diseases and falls within the scope of medical parasitology. Another important parasite in terms of prevalence and intensity of infection is the digenetic trematode L. paradoxum. Birds are infected by these flukes by pecking the eye stalks of infected snails Succinea putris [44,45] that contain sporocysts with metacercariae of these parasites, whose appearance lures feeding birds.
The differences between the helminth community structure of the Great tit and Eurasian blue tit, such as the larger number of helminth species in the Great tit, significantly different prevalence of helminths between the two species, significantly different abundance of helminths, and significantly different prevalence of L. paradoxum between the two host species, can be explained by the following factors: - The larger number of helminth species found in the Great tit was linked to the larger sample size; more than twice as many Great tits were examined as Eurasian blue tits. -Although both species prefer similar biotopes, the Great tit shows greater ecological tolerance regarding habitat conditions, which means that it may also inhabit poorer environments, e.g., coniferous forests or urban areas. The Eurasian blue tit is more closely associated with broadleaf forests and parks. A key to understanding the differences may be their feeding sites. The Great tit prefers feeding on the ground, while the Eurasian blue tit avoids foraging on the ground and prefers to penetrate tree crowns and shrubs. - The Great tit's higher tolerance for habitat conditions may result in more frequent contact with parasites present in the environment, as well as with their intermediate hosts, which may partly explain the significantly higher prevalence of helminths, mainly L. paradoxum (but also U. macrostomus), and the significantly higher mean abundance of helminths in the Great tit population than in the Eurasian blue tit population.

Presence of Parasites in the Geographical Distribution of C. careuleus and P. major
Our study characterised the diversity and abundance of internal parasites in the tits during the period of their autumn migration. However, the presence of the parasites can not be explained solely by the fact of bird migration since parasites may be present both at the nesting and wintering sites that largely overlap. The presence of parasites in tits does not stem from the migration itself but from their distribution in the habitat area of tits. However, due to the commonly observed clumped density of parasites in hosts and ecosystems, birds migrating between those areas can be invaded by diverse species and a number of pathogens. Therefore, birds that do not migrate and inhabit overlapping nesting and wintering areas can have different abundances and species diversity of parasites than birds that migrate. At this time point, the comparison of parasite populations for C. caeruleus and P. major is hindered due to a lack of detailed information on the parasites of tits during nesting and wintering periods. Among the parasites identified in our study, the high incidence of C. pavlovskyi is presumably related to the migration of birds. In 1971-1988, this species of nematode was identified in P. major nesting in Poland with a prevalence of 6% and a range of intensity 1-15 [46]. Regarding the number of parasites in hosts, expressed by mean intensity or mean abundance, the high values might result from environmental and physiological stress, but not only from the superinfective processes [47].

DIGENEA
The most common have been species of trematodes, followed by cestodes and nematodes. Data from outside Poland are primarily from the Czech Republic, Eastern Europe, the Asian part of Russia, Ukraine, Finland, and Slovakia [25,26,36,[48][49][50][51][52][53][54][55][56][57][58][59][60]. Bird migration plays a major role in the transmission of parasites between ecosystems, but this process is little known in songbirds, in contrast to wetland and water birds [61,62]. The range of most bird helminth species identified in this study is Palaearctic, and some of them are cosmopolitan. Migratory birds often are infected by new species of parasites in ecosystems located along the migration route and at the wintering grounds and transfer them to their breeding grounds, as well as from the breeding grounds to the wintering grounds. In autumn, many species of birds fly along the Baltic coast from their breeding grounds toward their wintering grounds. The association between avian blood parasites and the migration of birds is well known [4], but the association between helminths and these migratory birds is not. In autumn of the years 2008-2012, a total of 99,637 birds, including 38,917 Great tits and 7320 Eurasian blue tits, were captured and ringed at Operation Baltic stations on the southern coast of the Baltic Sea in Poland. Parasitological parameters determined in a research sample of these birds indirectly provide information on the occurrence of parasites in the entire migratory populations of the Eurasian blue tit and Great tit. The geographic distribution of parasites essentially overlaps with the range of their hosts (see: faunaeuropea.eu [63]). Parasitological parameters reflect the parasitic fauna of Great tits and Eurasian blue tits immediately before their migration, but these parameters can also be influenced by parasites acquired at stopover sites after the migration has begun. The Eurasian blue tit and Great tit migrate across short distances within Europe. The various parasite species found in the present study had previously been recorded in tits or other bird species in Eastern Europe (e.g., northeastern Poland) and in the Asian part of Russia, which may overlap with the breeding grounds of tits. They have also previously been recorded in Western and Southern Europe (e.g., northwestern Poland, the Czech Republic, Slovakia, Romania, Bulgaria, France, and Spain) [46], where seasonal migration of tits takes place or which serve as wintering grounds for these birds. The parasitological parameters of the tit parasites detected in the present study indicate that autumn migration is conducive mainly to the development of the populations of trematodes L. paradoxum and nematodes C. pavlovskyi, while other parasite species are rare in tits.
The tapeworm A. globata (Paruteniridae, Cyclophyllidea) was recorded in the present study for the first time in Poland. A. globata is widespread in many countries in Europe, including those along the migration route of tits, e.g., Germany, France, and Moldova [63]. Poland had previously been a gap on the map of Europe, which was filled owing to the research undertaken in this study. M. parinum is a cestode that is commonly recorded. It has previously been noted in Poland in Sturnus vulgaris [38]. The trematode L. paradoxum is a parasite of passerines and waders in Europe [64]. In Poland, this trematode has previously been recorded in Turdus merula, T. philomelos, C. caeruleus, and P. major, as well as in intermediate hosts, in practically all of Poland, including the migration route of tits from northeastern to northwestern Poland, together with the Baltic coast [38,49,50]. Outside of Poland, it has been recorded in Germany, Great Britain, Denmark, Finland, Moldova, Ukraine, Norway, Sweden, and the Czech Republic [45,48,63]. As indicated in the abovementioned works, trematodes of L. paradoxum are commonly present in Europe, including Poland. Thus, the identifications of L. paradoxum in migrating tits, both in the earlier studies and in our work, do not indicate that the migration of birds is the factor that influences infection of L. paradoxum but only shows that parasites are present in the birds during their migration.
U. macrostomus is a parasite of passerines in the Holarctic region. In Poland, it is noted in numerous bird species on the Baltic Sea coast as well as in the south of the country [38]. Outside of Poland, it has been recorded in many European countries, e.g., Moldova, Germany, France, Norway, and the Czech Republic [48,63]. P. maculosus is a trematode with broad host specificity in the Holarctic region. It is a frequent parasite of birds of several orders, including Passeriformes. It has also been noted in India, the Philippines, and Australia (according to Sitko et al. [48]). In Poland, it has been recorded in several species of birds on the Baltic Sea coast and in other parts of the country [38]. In Europe, it has been found in Lithuania, Moldova, Germany, and France [63]. The nematode C. pavlovskyi has Holarctic distribution and is known from Europe, Asia, and North America [46]. In Poland, it has previously been found in several Turdus species and in Parus major on the Baltic coast, as well as in southern Poland [40,46]. Outside of Poland, its presence has been noted in the northeast in the Kaliningrad Region, Latvia, France, and Spain [63]. C. pavlovskyi has also been recorded in birds in Ukraine [42]. The presence of C. triedens was previously reported in Europe among Passeriformes and birds of other orders, as well as in North America, Central America, and Asia. This ringworm had been recorded in Poland in several species of birds, including the Great tit, and outside of Poland in France and Spain [26]. The presence of D. henryi was previously recorded in Europe in Passeriformes, including Great tit, and in birds of other orders-in Poland, but also in Asia and Africa. In Europe, it has been recorded in birds of various orders in France, Romania, and Bulgaria [46]. The nematode D. obtusa has been reported in northeastern and central Poland [40]. In Europe, the presence of D. obtusa has been noted mainly in the northeast (Kaliningrad, Moldova) and south (Bulgaria, Italy, Ukraine), but also in the north (Netherlands) [63].

Similarities and Differences in Juvenile and Adult Tits and in Males and Females
The number of parasite species in adult Eurasian blue tits is greater than in juveniles, while in the Great tit, it is the same in adults and juveniles. In adult birds, an infection can take place at both the breeding grounds and the wintering grounds, which for both species cover northwestern Poland, northern Germany, the Netherlands, and Belgium, and in the case of Great tit, also northeastern France. Therefore, the parasite fauna of adult birds may be partially mixed. Helminths occurring in juvenile birds migrating in autumn for the first time will mainly represent local parasites present in the host breeding grounds, but it cannot be ruled out that infection with these parasites could take place during autumn migration. Most of the differences between the parameters of infection of juvenile and adult tits were not statistically significant, with the exception of the prevalence of cestodes, which was significantly higher in adult Great tits than in juveniles. Differences in infection depending on the age of the host are well documented in the literature for various animal species e.g., [62]. The higher prevalence of tapeworms in adult Great tits is linked to the more frequent share of the intermediate hosts of these parasites in their diet than in juvenile birds.
The number of parasite species was higher in females of both tit species than in males ( Table 3), but there were no significant differences between the parameters of infection of females and males in the two species of tit. The results of studies by various authors on the relationship between infection and the sex of the host are inconclusive. The relationship between parasite infection and host sex may depend on other environmental factors [65,66]. Experimental research by Richner et al. [65] showed that in males from Great tit families in which the number of nestlings in the nests was experimentally increased, the rate of infection with malaria parasites was twice that of males from families in which the number of nestlings was not increased. This was explained by the fact that the chicks were fed by the males, not the females [65]. Studies of Blackbird Turdus merula have shown the influence of an anthropogenic environment on differences in helminth infection of males and females. Differences between the infection parameters of males and females were observed in an urban Blackbird population but not in a forest population [62].

Conclusions
The new information provided in this study has increased our knowledge of the transfer of helminths during the migration of birds in central Europe, and the Eurasian blue tit and the Great tit were shown to be important carriers of filarial nematodes Cardiofilaria pavlovskyi, Diplotriaena henryi, and Diplotriaena obtusa. This research is necessitated by gaps in current knowledge of changes taking place in the population of helminth species in the context of global environmental changes, especially climate change. Similarities and differences in helminth communities of the Great tit and Eurasian blue tit were demonstrated, with a greater spread of Leucochloridium paradoxum observed in the population of the Great tit than in that of the Eurasian blue tit. There is a need to monitor the size of communities and populations of helminth species such as L. paradoxum, whose high intensity of infection may affect the health and body condition of birds. Based on the results of the study, we recommend increased research on the occurrence of parasitic helminths in migratory birds, examination of birds for filarial nematodes, and studies of populations of different species of Leucochloridium in the environment.  Data Availability Statement: The raw data are available from the authors. The sequences from the study were uploaded to National Center for Biotechnology Information, GenBank accession MK882500-MK882508.