Introduction

Waterfowls are well-known indicators of wetland quality. A powerful tool based on this characteristic is the so-called 1% criterion: any site which regularly holds 1% or more of a water bird population qualifies as a wetland of international importance under the Ramsar Convention on Wetlands. The 1% criterion has been adopted by the European Union to identify Special Protection Areas (SPAs) under the Birds Directive and by BirdLife International to identify Important Bird Areas (IBAs) in wetlands throughout the world (Delany 2011).

Lake Ladoga in Northwestern Russia is particularly important for waterfowls because the White Sea-Baltic branch of the East Atlantic Flyway passes across it. In total approximately 70 species of birds use the lake and the area around it for temporary respite during migrations or nesting (Noskov 1997; Noskov et al. 2016). Wetlands of the Olonets plain (60048′-61004′ and 32046′-33014′) situated at the south-eastern coast of Lake Ladoga are important stopover sites for geese and many other species of birds on their spring and autumn migrations, and require protection. The Olonets wildlife area of national significance borders the Nizhnesvirsky State Nature Reserve situated in the Svir River Delta (Zimin et al. 1997).

There are excellent conditions for waterfowl to nest and feed in the East Ladoga region, which is due to the combination of several factors. Grass-and-sphagnum mesotrophic bogs are common in the region, the degree of bogginess varying from 20 to 50% (Elina et al. 2005). In addition to Lake Ladoga itself, there are numerous small lakes located in the forests and open bog land in the region. Vast shallow areas on the lake Ladoga’s coast have a high biological productivity and offer considerable natural resources for foraging birds (Noskov 1997; Zimin et al. 1997).

Parasites are important co-evolving components of the natural environment, with close ties to their hosts. Having a detailed knowledge of the species composition of waterfowl parasites and their distribution in host populations is important for predicting the risk of outbreaks of infectious diseases. Additionally, the parasitic fauna of birds may serve as markers of migration pathways and feeding habits with a number of studies having already been carried out on birds in the he birds of Karelia region (Lebedeva et al. 2013, 2015, 2017, 2020; Yakovleva et al. 2020; etc.).

The Eurasian coot Fulica atra Linnaeus, 1758 (Rallidae Rafinesque, 1815) is a common and abundant waterfowl species in southern Karelia. Until the mid-twentieth century the Eurasian coot was rare in North western Russia but its numbers have been increasing in recent decades with its distribution gradually spreading northwards and eastwards. According to the results of bird ringing, coots from Northwestern Russia winter in the UK, Denmark, Italy, the Netherlands, France, Sweden and Switzerland (Noskov et al. 2016).

The parasitic fauna of F. atra has been extensively studied in different parts of Eurasia (Pukhov 1939; Bashkirova 1941; Ginetsinskaya 1952; Bykhovskaya-Pavlovskaya 1953; Shakhtahtinskaya 1959; Pavlov 1960, 1962, 1966; Sergienko 1972; Smogorzhevskaya 1976; Pojmanska 1982; Nekrasov 2000; Semenova and Ivanov 2005; Sitko et al. 2006; Gherman et al. 2008; Fedorovich et al. 2010; Sitko and Okulewicz 2010; Sitko 2011; Mahmudova 2012; Zhang et al. 2012; Ivanov et al. 2013; Penkina and Okolelov 2013; Królaczyk et al. 2018, 2020). However, there is no information about the parasite fauna of the Eurasian coot along the coast of Lake Ladoga.

The aim of this study was to explore the helminth fauna of Eurasian coots on Lake Ladoga’s coast and to investigate some aspects of the biology of these birds potentially affecting their parasites.

Materials and Methods

Sampling

The coots were shot by licensed hunters on the south-eastern coast of Lake Ladoga (61°12′N, 32°54′E, Russia). In total, 22 specimens (14 males and 8 females) of F. atra were dissected in the autumn of 2011–2014. Ten of the males and five of the females were young birds.

Age and sex of the coots was determined following standard procedures (Kurochkin and Koshelev 1987). Young birds were distinguished from the adult birds based on the presence of the bursa of Fabricius. In the process of dissection, the remains of food in the intestinal tract of the birds were also examined.

The parasitological examination of the birds was conducted according to Dubinina (1971). All organs and the intestinal tract of the coots were examined for infection with helminths. All the parasites found were localized in the intestinal tract. No parasites were recorded from the adipose tissue, muscles, internal organs and glands (windpipe, lungs, heart, kidneys, liver and gall bladder, the pancreas and the sex glands).

The parasites were fixed and stored in 96% ethanol. Cestodes and trematodes were stained with acetocarmine and mounted in Canada balsam. The nematodes were cleared in 80% lactic acid. Acanthocephalans were mounted in Berlese chloral gum (Dubinina 1971).

Morphometric methods based on systematic keys were used for identification (Filimonova 1985; Sonin 1985; Khokhlova 1986; Tolkacheva 1991; Jones 2008; Lotz and Font 2008; Królaczyk et al. 2020). A morphological study of parasites was carried using the Olympus CX-41 video system (Olympus Corporation, Tokyo, Japan) and the Levenhuk C1400 NG, LevenhukToupView software, V.3.5 (equipment of Core Facility of the Karelian Research Centre of the Russian Academy of Sciences). The systematic list of the parasites is given according to the World Register of Marine Species (http://www.marinespecies.org/).

DNA Extraction, Amplification, Sequencing, and Phylogenetic Analyses

Genomic DNA was extracted from two immature and two subgravid specimens of the genus Prosthogonimus Lühe, 1899 using DNA-Extran kits (Synthol, Moscow). To obtain the 28S rRNA gene sequences, the trematode-specific forward primer DIG12 (5′ – AAG CAT ATC ACT AAG CGG – 3’and reverse primer 1500R (5′ – GCT ATC CTG AGG GAA ACT TCG – 3′) of Olson et al. (2003) were used. The thermal cycling parameters were as follows: initial denaturation at 95 °C (3 min); 35 cycles of 20 s at 95 °C; 20 s at 56 °C; 120 s at 72 °C; 5 min at 72 °C for the final extension. The 28S rRNA gene fragments were amplified using the BIO-RAD C1000 Thermal Cycler. PCR was performed in a total volume of 20 μl. Amplicons were purified using the Cleanup Mini Purification Kit (Eurogene) and sequenced directly using PCR primers and internal primers 300F (5′ – CAA GTA CCG TGA GGG AAA GTT G – 3′) and ECD2 (5′ – CTT GGT CCG TGT TTC AAG ACG GG – 3′). DNA sequencing was performed using the ABI PRISM® BigDye™ Terminator v. 3.1 reagent kit followed by analysis of the reaction products on a Genetic Analyzer ABI PRISM 3130 automatic DNA sequencer (Applied Biosystems, USA) at the Core Facilities Centre “Taxon” (Zoological Institute, St. Petersburg, Russia).

The four sequences of the partial 28S rRNA gene (1253 bp) were aligned with ClustalW implemented in MEGA v. 10 (Kumar et al. 2018) and deposited in GenBank under accession numbers MW114967– MW114970.

A standard BLASTn search of closely related species in GenBank was conducted using ClustalW in MEGA v. 10. The partial 28S rRNA gene sequences generated in the study were aligned against representatives of the order Microphalloidea (Supplementary Table S1). Plagiorchis koreanus Ogata, 1938 (family Plagiorchiidae Lühe, 1901) was used as an outgroup. The length of the alignment is 1260 bp. Phylogenies were reconstructed using maximum likelihood (ML) and Bayesian inference analysis (BI) approaches implemented in MEGA 6.0 and MrBayes software (ver. 3.2.3) (Ronquist et al. 2012), respectively. In addition, jModelTest jModelTest 2.1.2 (Darriba et al. 2012) was used to estimate the best nucleotide substitution model for the dataset. In both analyses, the general time-reversible model GTR + G + I dataset based on AIC was used. Branch support was estimated by bootstrap analyses with 1000 replicates, and BI was conducted with 10,000,000 generations. FigTree ver. 1.4 software (Rambaut 2012) was used to visualize the trees.

Statistical Analysis

The data on the parasites were interrogated, and the prevalence (P) and the mean abundance (M) were calculated according to Bush et al. (1997) as follows:

$$P=\frac{Ni}{N}\bullet 100$$
$$M=\frac{\sum n}{N}$$

Statistical analysis of the infection characteristics and the distribution of the parasites was performed using Quantitative Parasitology software (Rózsa et al. 2000; Reiczigel et al. 2019).

The accumulation curve of species richness of the parasites (rarefication curve) with the increasing number of studied birds was based on the data on the absence/presence of the parasite species; the number of parasite individuals of a given species was not taken into account. The analytical solution was implemented as “Mao’s tau” with the standard deviation (Colwell et al. 2004). On the graph, the standard errors were converted into 95% confidence intervals. The curve was constructed and analysed using the PAST Ver. 3.19 software (Hammer et al. 2001).

The degree of similarity of the helminth fauna was measured by the Jaccard index. Cluster analysis was performed (Jaccard index, UPGMA Algorithm) and the bootstrap dendrogram was constructed also using PAST Ver. 3.19 software (Hammer et al. 2001).

Results

Feeding

All the birds examined in our study had fragments of plant food in the intestinal tract, including vegetative parts of plants, and seeds. Animal food was found in 46% of the birds; more specifically, the remains of insects were found in 23% of the birds and the remains of molluscs in 32% of the birds. Animal food made up 53.3% of the diet of young coots. In contrast, only 28.6% of adult coots had the remains of molluscs, in addition to plant food, in their intestinal tract.

Parasite Fauna

The intestinal tract of the coots examined was found to contain 11 helminth species: four trematodes, four cestodes, two acanthocephalans and a nematode (Table 1). The highest prevalence was registered for cestodes (95%) and trematodes (86%). The prevalence of acanthocephalans and nematodes in the coots was 23% and 5%, respectively. The results of our study indicate that the parasitic fauna of F. atra from Lake Ladoga coast are mainly represented by species with a complex life cycle.

Table 1 Helminths of the Eurasian coot (Fulica atra Linnaeus, 1758) from Northwestern Russia
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Acanthocephalans were represented by Filicollis anatis (Schrank, 1788) Lühe, 1911 and Polymorphus minutus (Zeder, 1800) Lühe, 1911. Both these species had a low prevalence.

The cestodes, isolated from avian samples, were represented by four species of the genus Diorchis Clerc, 1903 (Hymenolepididae Ariola, 1899), which were found in 21 out of the 22 examined birds. The most common species were D. ransomi Schultz, 1940 and D. inflata (Rudolphi, 1819), while D. brevis Rybicka, 1957 was much less common and D. stefanskii Czaplinski, 1956 was only found in a single host individual (Table 1).

The four trematode species isolated from coots belonged to four families: Psilostomidae Looss, 1900 (Psilotrema spiculigerum), Notocotylidae Lühe, 1909 (Notocotylus pacifera), Lecithodendriidae Lühe, 1901 (Leyogonimus polyoon) and Prosthogonimidae Lühe, 1909 (Prosthogonimus ovatus).

Trematodes Leyogonimus polyoon (Braun, 1902) Ginetsinskaya, 1947 were not numerous. All the worms isolated from samples were mature, with their uterus filled with eggs. A single individual of the trematode Psilotrema spiculigerum (Mühling, 1898) Odhner, 1913 was also found.

After dissecting all the samples it was found that Notocotylus pacifera (Noble, 1933) was the most common trematode (Table 1). Both mature and immature parasites were found in young and adult birds.

Another numerous trematode species was P. ovatus (Rudolphi, 1803) Lühe, 1899 (Fig. 1A, B) which was recorded only in young birds where fully-gravid, sub-gravid and immature specimens were found. To verify the species affiliation of sub-gravid and immature worms, we used the 28S rRNA gene as a molecular marker. Molecular data confirmed that these specimens belonged to P. ovatus (Fig. 2).

Fig. 1
figure 1

Subgravid specimens of Prosthogonimus ovatus: A. Dorsal view. B. Ventral view. Scale bar 0.5 mm

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Fig. 2
figure 2

Phylogenetic position of sugravid Prosthogonimus ovatus based on the analysis of 28S rDNA partial sequences using ML and BI algorithms. Nodal support: BI/ML. Newly obtained sequences are shown in bold

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A single specimen of the nematode Quasiamidostomum fulicae (Rudolphi, 1819) Lomakin, 1991 was found inside a cyst-like structure under the cuticle of the gizzard of a young bird (Fig. 3). The parasite was found to be a female at the early fifth-stage of development. The body of the Q. fulicae specimen was 4.46 mm long and 0.12 mm wide. The buccal cavity was well-developed and thick-walled; its bottom supported a single tooth 0.01 mm in length (Fig. 3a). The tail was 0.115 mm long, tapered posteriorly (Fig. 3b).

Fig. 3
figure 3

Morphology of subadult female of Quasiamidostomum fulicae from coot of Lake Ladoga: A. Anterior region. B. Posterior region. Scale bar 0.1 mm

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The species composition of the parasites were found to be different in young and adult coots (Table 1). While young individuals harboured 11 helminth species, only four species (F. anatis, D. brevis, D. ransomi and N. pacifera) were registered in adult coots. However, comparing the infection indices of the young and the adult birds (Table 1), we found statistically significant differences only in the prevalence of P. ovatus (p = 0.005). There was no significant statistical differences (p > 0.05) found between the prevalence and the mean abundance for each parasite species in male and female coots.

Four of the helminth species found in F. atra of Lake Ladoga region (36% of the species composition) were noted only once (in a single bird individual). The accumulation curve of the helminth species richness agrees with the equation of the power function (Fig. 4).

Fig. 4
figure 4

Accumulation curve of helminth species based on the parasitological survey of the Eurasian coot (22 ind.), with a 95% confidence interval (2δ)

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Cluster analysis of parasite species composition from different regions (Fig. 5) showed that they were divided into two clusters: regions with low (Baikal Lake basin, Armenia, Karelia) and high (Czech Republic and Slovak Republic, the Ukraine, Volga drainage basin) helminth diversity. When our data were compared with those from other regions, the similarity of helminth species composition (Supplementary Table S2) was low. Pairs’ Jaccard Index values varied from 0 (Romania) to 0.25 (Armenia).

Fig. 5
figure 5

Dendrogram of the cluster analysis (Jaccard index, UPGMA Algorithms) of the parasite fauna of Fulica atra from 7 regions: 1 – Baikal Lake basin, 2 – Armenia, 3 – Karelia (our data), 4 – Czech Republic and Slovak Republic, 5 – Ukraine, 6 – Volga drainage basin, 7 – Romania. The percentage of replicates where each node is supported is given (bootstrap replicates = 500)

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Discussion

The Eurasian coot inhabits various water bodies, where it resides in shallow areas with abundant aquatic vegetation. The diet of the Eurasian coot mainly consists of shoots and seeds of various aquatic plants, algae, sedge, pondweed and duckweed. Animal food such as molluscs, insects and crustaceans makes up not more than 10% of its diet (Brisbin and Mowbray 2002; Zimin and Ivanter 2002). Current evidence would suggest that young coots feed only on animal food, especially for the first two weeks of their life, and then gradually switch to feeding on vegetation (Golovan et al. 2011). Our analysis of the content of the intestinal tract of the coots revealed that young birds had a more diverse diet than older ones.

The parasitic fauna of F. atra from Lake Ladoga’s southeastern coast is mainly represented by species with a complex life cycle. It is likely that the coots get infected with these parasites while feeding on different invertebrates or vegetation.

The intermediate host of the acanthocephalan F. anatis is the water louse Asellus aquaticus Linnaeus, 1758 (Kotelnikov 1959), while that of P. minutus is the freshwater shrimp Gammarus lacustris (Khokhlova 1986). Both of these crustacean species form part of the benthos, and the birds are possibly infected when they fed, including the infected crustaceans, from the bottom of the shallows. A low prevalence of acanthocephalans species in coots (Table 1) indicates that they feed on benthos rarely, or even accidentally.

The development of cestodes of the genus Diorchis is associated with crustaceans from the groups Copepoda, Ostracoda, Amphipoda and Cyclopoida (Czaplinski and Szelenbaum 1974; Rybicka 1957; Tolkacheva 1991). These organisms are numerous in the zooplankton in shallow areas of Lake Ladoga with an abundance of 98,000–124,000 ind./m3 and a biomass of 0.85–1.65 g/m3 (Kurashov et al. 2018). The coots probably primarily ingest crustaceans together with vegetation, which could possibly explain a high prevalence of cestode species.

The trematodes N. pacifera and P. spiculigerum showed significant differences in invasion rates (Table 1), although they have a similar life cycle. Cercariae of both species develop within gastropods, then leave the host and transform into the adolescaria stage, encysting on aquatic vegetation, underwater objects or even shells of molluscs, which are then eaten by birds. The snail Physa fontinalis (Linnaeus, 1758) is the intermediate host of N. pacifera (Odening 1964a, 1964b, 1966), while snails Bithynia leachi (Sheppard, 1823) and Bithynia tentaculata (Linnaeus, 1758) are the intermediate hosts of P. spiculigerum (Mathias 1925; Belyakova 1978). B. tentaculata and P. fontinalis have different distribution around the Lake Ladoga water area (Barbashova and Kurashov 2011; Belyakov 2013; Kurashov et al. 2013). B. tentaculata inhabits only the coastal zone of Lake Ladoga while P. fontinalis occurs in the algae thickets of the lake and its tributaries. We can therefore suggest that the coots mainly fed on algae in the biotopes, where P. fontinalis snails prevailed. Both mature and immature specimens of N. pacifera were numerous, indicating a stable infection of the host. This could be one of the reasons for the high infestation of coots by N. pacifera. Another possible reason behind the different infection of coots with trematodes N. pacifera and P. spiculigerum is the different specificity of these parasites for F. atra. N. pacifera is an obligate parasite of rails, including coots, and cannot develop in other birds (Filimonova 1985). In contrast, P. spiculigerum can parasitize many birds from the families Anseranatidae, Anatidae and Rallidae as well as some small mammals (Skryabin 1947; Smogorzhevskaya 1976; Sonin 1985).

The coots potentially get infected by P. ovatus and L. polyoon when feeding on aquatic larvae of dragonflies and caddisflies, which are the second intermediate hosts of these parasites (Cole and Friend 1999; Cole 2001; Heneberg et al. 2015). Adult coots do not feed on insects, which explains the fact that both these trematodes were found only in young individuals (Table 1). The prevalence of L. polyoon in coots was much lower than that of P. ovatus. This may be associated with a different range of the first and the second intermediate hosts of these trematodes in Lake Ladoga as well as their different availability to the coots.

The nematode Q. fulicae was identified based on the diagnostic characters developed by Lomakin (1991). Based on the presence of a broad thick-walled buccal capsule and a single tooth, as well as the host species, it belongs to the genus Quasiamidostomum, of which Q. fulicae is the only species. As the specimen had a well-developed armed buccal capsule, it was likely a sub-adult female at the early fifth-stage of development.

The nature of the cyst in which this nematode was found remains obscure. A similar cyst has been noted in Epomidiostomum crami (Wetzel, 1931) but nothing like this has been recorded in Amidostomatinae Travassos, 1919 (Tuggle 1982). The life cycle of Q. fulicae nematodes is direct, the third stage larvae being directly ingested by birds (Czaplinski 1962; Baruš 1964). A low infestation of the coots by these nematodes in the current study may be associated with low air temperatures in the Lake Ladoga region in late summer and autumn (10–18 С0), because their “larvae required 5 days of free-living existence at 18 to 20 С0 to become infective” (Leiby and Olsen 1965, p. 39).

Most of the 11 helminth species found in the F. atra from the Lake Ladoga coast in this study are common parasites of other waterfowl species in the Holarctic and the Neotropical region. Only a few parasites are specific to the Fulica spp. One of them is the trematode N. pacifera, which is a specialized parasite of coots in the Holarctic (Filimonova 1985; Kulišić et al. 2004; Hannon et al. 2016). In the Neotropical region, however, these trematodes also infect other waterfowl (Padilla-Aguilar et al. 2020). Similarly, the nematode Q. fulicae has been reported almost exclusively from coots, being much rarer in other waterfowl in the Palearctic (Królaczyk et al. 2020). The trematode L. polyoon is also a specific parasite of the Eurasian coot (Sitko et al. 2006; Kanarek et al. 2017). Other helminths can parasitize many waterfowl species. The trematode P. spiculigerum was also found in the Eurasian wigeon (Mareca penelope Linnaeus, 1758) in Karelia (Yakovleva 2013). The cestode D. ransomi was recorded in Karelia in geese while D. stefanskii was registered in the mallard (Lebedeva et al. 2014; Yakovleva et al. 2018).

The parasitic fauna of F. atra of Eurasia comprises 117 species: four acanthocephalans, 25 nematodes, 21 cestodes and 67 trematodes (Pukhov 1939; Bashkirova 1941; Shakhtahtinskaya 1959; Pavlov 1960, 1962, 1966; Pavlov and Sergeeva 1961; Bykhovskaya-Pavlovskaya 1962; Sergienko 1972; Smogorzhevskaya 1976; Borgarenko 1981, 1984, 1990; Pojmanska 1982; Maksimova 1989; Nekrasov 2000; Movsesyan et al. 2004, 2006, 2017; Semenova and Ivanov 2005; Sitko et al. 2006; Gherman et al. 2008; Fedorovich et al. 2010; Sitko and Okulewicz 2010; Sitko 2011; Mahmudova 2012; Zhang et al. 2012; Ivanov et al. 2013; Penkina and Okolelov 2013; Supplementary Table S2). In some localities, however, only certain groups of parasites infecting F. atra have been studied (Pojmanska 1982; Zhang et al. 2012; Królaczyk et al. 2018, 2020; etc).

Parasite fauna of coots in the Palearctic is best studied in the Czech Republic, the Slovak Republic, the Ukraine, Romania and Russia (Volga drainage basin, Baikal Lake basin). Among them, the greatest diversity of parasite species is observed in the Czech Republic and the Slovak Republic (43 species). Parasite species numbers recorded elsewhere are Ukraine (38 species), Southern Russia (the Volga drainage basin) (30 species), Romania (4 species), Baikal Lake basin (11 species), and Armenia (19 species). Out of 11 species of parasites found in Karelia in coots, 9 were also found in the Czech Republic and the Slovak Republic and 8 in Ukraine. Prosthogonimus ovatus, Diorchis brevis, D. inflata, D. ransomi, Filicollis anatis, Polymorphus minutus were widespread in all territories except Romania. The differences in the composition of the coot helminth fauna in different territories may be associated with methodological differences between the studies including a different number of birds examined, a different number of locations in each territory, different seasons, etc.

The accumulation curve of the helminth species richness agrees with the power function, indicating its non-asymptotic character (Dove and Cribb 2006). This means that the examination of 22 individuals did not reveal the full species richness of the parasite component community of this coot population. The habitat of the host also plays an important role in the host-parasite relationships (Gower 1939). An examination of birds from a greater number of different habitats may reveal a higher diversity of the parasite communities in the region. The differences in the species composition of coot’s parasites in different regions may be determined by differences in the feeding patterns. Ecological characteristics of the birds, especially their diet, are known to be variable depending on the habitat and other biological factors.

Conclusion

We presented the first data on helminths parasitizing the Eurasian coot in the Lake Ladoga region of Northwestern Russia, lying at the White Sea-Baltic branch of the East Atlantic Flyway. In summary, the list of parasites of Eurasian coot F. atra from the Lake Ladoga coast is likely to be extended after further research, involving a larger number of birds and conducted in a different season, e.g. in spring.