Taxonomic status of Cyathostoma nematodes (Nematoda: Syngaminae) parasitizing respiratory tracts of birds of prey and owls in Europe and North America: how many species are there?
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Introduction
The subfamily Syngaminae Baylis & Daubney, 1926 comprising parasites of respiratory tracts of birds (genera Boydinema, Cyathostoma, Syngamus) and mammals (genera Mammomonogamus and Rodentogamus). The largest (more than 20 species) is genus Cyathostoma, a taxa with a long and confused history. This genus was established by Blanchard (1849) for Cyathostoma lari, species described from the orbital cavity of the black-headed gull Chroicephalus ridibundus. Over a period of years, the validity of Cyathostoma was widely discussed: some authors recognized Cyathostoma as a synonym of Syngamus (e.g., Skryabin, 1915; Yamaguti, 1961), but others accepted this taxon as correct. T However, the definition and taxonomic position of Cyathostoma has been modified several times in relation to different evaluation of morphological features (e.g., Chapin, 1925; Ryzhikov, 1949; Hovorka & Macko, 1959; Turemuratov, 1963; Ryzhikov, 1967; Lengy, 1969; Ali, 1970; Baruš & Tenora, 1972). In the most recent system of Syngaminae proposed by Lichtenfels (1980) the genus Cyathostoma is divided into two subgenera in relation to the structure of copulatory bursa and spiculae length: Cyathostoma (Cyathostoma) (Blanchard, 1849), in which the dorsal ray extends beyond the end of the copulatory bursa to form characteristic thorn-like projections and spicules that measure 0.08 – 0.4 mm, and Cyathostoma (Hovorkonema) Turemuratov, 1963 in which the dorsal ray does not extend beyond the end of the copulatory bursa and spicules measure 0.45 – 0.8 mm. In the systems of Syngamidae created by Ryzhikov (1967) and Baruš and Tenora (1972)Cyathostoma and Hovorkonema are treated as independent genera.
Occurrence of Cyathostoma nematodes have been reported in a variety species of birds from several orders (Kanarek et al., 2013). Indisputably, Cyathostoma infection has great significance as a potential pathological factor in poultry production and many aspects of conservation of vulnerable or endangered avian species (for details see e.g., Fernando & Barta, 2008 and references therein). Usually, occurrence of these nematodes is subclinical, but heavy infections are often associated with bacterial and/or fungal infections. Several reports of morbidity and mortality of birds, particularly birds of prey and owls caused by infection of Cyathostoma are available (e.g., Hunter et al., 1993; Lavoie et al., 1999; Krone et al., 2007; Vaughan-Higgins et al., 2013). Thus, understanding of all aspects of ecology and taxonomy of these syngamid nematodes has great practical importance for veterinary and conservation reasons in general. Currently, four species of these nematodes have been recorded from birds of prey (Accipitriformes, Falconiformes) and owls (Strigiformes): C. (Hovorkonema) americanaChapin, 1925; C. (Hovorkonema) brodskiiSultanov, 1946; C. (Hovorkonema) variegatum (Creplin, 1849) and C. (Cyathostoma) lariBlanchard, 1849. Moreover, several reports on the occurrence of Cyathostoma nematodes determined only to the genus level in birds of prey and owls are available (e.g., Mumcuoglu & Müller, 1974; Hunter et al.,1993; Lavoie et al., 1999; Vaughan-Higgins et al., 2013). Among the mentioned species, C. (Cyathostoma) lari is a typical parasite of gulls, only occasionally reported from birds of prey (Simpson & Harris, 1992). However, when delimitation of C. (Cyathostoma) lari from other mentioned C. (Hovorkonema) species recorded in birds of prey and owls regarding characteristic habitat (C. (Cyathostoma) lari occurred in nasal and orbital cavities, while other species are detected in tracheae, bronchii and air sacs) and morphological features typical for nominal subgenus Cyathostoma (structure of copulatory bursa, spiculae length) are rather trivial, thus morphological boundaries between closely related C. (Hovorkonema) americana, C. (Hovorkonema) brodskii and C. (Hovorkonema) variegatum are still contentious. Moreover, several revisions and doubtful synonimisations (e.g., Vogel, 1928; Ali, 1970; Ryzhikov, 1980) increased confusion. In our opinion, the crucial problem in contemporary taxonomy of Cyathostoma (including parasites of raptors) is poorly recognized morphological variability among/within species and disputable host specificity, resulting with large number of species with questionable validity. Thus, final confirmation of the validity of several C. (Hovorkonema) species, irrespective of evaluation of different morphological, morphometric and ecological data, required molecular methods. Unfortunately, up to the present, analyses of taxonomic and phylogenetic relationships among subfamily Syngaminae based on molecular markers are very scarce (Krone et al., 2007; Kanarek et al., 2013). So far, only Krone et al. (2007) analyzed molecular status and genetic diversity of nematode identified as Hovorkonema variegatum, collected from respiratory tracts of several birds species (including birds of prey) from Germany and based on the divergence of the compared ITS-2 sequences recognized existence of two distict groups of Cyathostoma (Hovorkonema) variegatum nematodes. On this base, the autors speculated about a occurrence of subspecies or a cryptic species, which are morphologically indistinguishable. In light of this, further studies regarding validity and molecular diversity of these nematodes occurring in birds of prey and owls are required.
Therefore, the main objective of the presented research was molecular identification and analyzes of SSU ribosomal gene diversity of C. (Hovorkonema) nematodes, collected from respiratory tracts of several species of raptors from Central Europe and North America. The obtained data are presented and discussed, and additionally, a hypothesis regarding validity of the avian C. (Hovorkonema) species is suggested.
Material and Methods
Sampling protocols, necropsy procedures and morphological identification
In spring of 2014, two subadult snowy owls Bubo scandiacus were found dead in Rhode Island, USA, and were transported to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL). Necropsy revealed the presence of medium-sized, red nematodes in the air sacs and on the surface of the lung, identified visually as Cyathostoma sp. Worms were washed in tap water, fixed and stored in 70 % ethanol. Additionally, during standard helminthological analysis of carcasses of birds of prey and owls, obtained from the Rescue Station for Animals, Bartošovice na Moravě, Czech Republic, nematodes initially identified as Cyathostoma sp. were found in air sacs of single Eurasian sparrowhawk Accipiter nisus, long-eared owl Asiootus and Eurasian eagle-owl Bubo bubo. The nematodes were washed in tap water, fixed in 70 % ethanol and stored in the same medium for further examination.
Detailed analysis of sampled material, showed that all collected nematodes were adult females. Unfortunately, the lack of males prevented precise morphological determination to the species level. Voucher specimens have been deposited in the Polish Collection of Parasitic Helminthes, Museum of Natural History, Wrocław University, Coll. No. 154285 (specimen ex Asia outs),154286 (specimen ex Bubo bubo), 154287 (specimen ex Accipiter nissus) and154288 (specimens ex Bubo scandiacus).
Molecular analysis
Material for the molecular analysis consisting of fragments (ca. 1 cm long) of the nematodes obtained from four host species (Accipiter nissus, Asio otus, Bubo bubo, B. scandiacus) was fixed as above and stored in 70 % ethanol. Total genomic DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s protocol. Amplification of ITS1-5.8S rDNA-ITS2 fragments was done using the following primers (van der Auwera et al., 1994) – NLF/NLR (5’-TTTGyACACACCGCCCGTCG-3’/5’-ATATGCTTAArTTCAGCGGGT-3’) in the following thermocycling conditions: 95 °C/3 min. – initial denaturation; 95 °C/30 sec., 48 °C/30 sec., 72 °C/45 sec. – 40 cycles; 72 °C/7 min – final extension. The PCR reaction (25 μl) was obtained: 4 μl of genomic DNA, 10mM Tris-HCl, 50mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 150 pmol of each primer and 2 units of Taq polymerase (EurX, Gdańsk, Poland). The amplification product was purified using QIAquick PCR purification kit (Qiagen, Düsseldorf, Germany) and sequenced in both directions (Genomed S.A., Poland). The obtained sequences were deposited in GenBank under following accession numbers KR349495-KR349498.
In order to elucidate any homologies with previously deposited sequences in GenBank, a BLAST search (http://blast.ncbi.nlm.nih. gov/Blast.cgi) was conducted. The multiple alignment was done by the use of CLUSTAL W using MEGA 5.0 package (Tamura et al., 2011). Phylogenetic analysis of C. (Hovorkonema) was inferred using the Maximum Likelihood method.
Results
As a result of molecular analysis, four 929 – 934 bp long sequences of ITS complex (ITS1, 5.8S rDNA and ITS2) for C. (Hovorkonema) sp. were obtained. In the overlapping fragment (930 bp) all four sequences were identical and no genetic diversity was observed. In addition, the comparison of our sequences with those deposited in Gen Bank showed none (AY702699) or a single nucleotide difference (DQ679968) with the sequences described as “Hovorkonema variegatum”. At the same time, our sequences showed quite large genetic variability with the two latter sequences of H. variegatum deposited under accession numbers: AY702698 (38 sites out of 297 bp long alignment – 12.8 %) and DQ679967 (36/297 bp – 12.1 %). The ML phylogenetic analysis (based on ITS2) performed for our sequences and the sequences of H. variegatum deposited in Gen Bank showed the presence of two clades, one including six identical or almost identical (1 nucleotide difference) sequences of syngamid nematodes from birds of prey (KR349495-KR349495) and sequences previously deposited by Krone et al. (2007) (AY702699 and DQ679968) (Fig. 1). The second clade comprises two sequences (AY702698 and DQ679967) of H. variegatum.
Fig. 1.
Phylogenetic analysis of ITS1 and ITS2 sequences performed using Maximum Likelihood method, after fitting the best model (Tamura 3-parameter); bootstrap support calculated on basis of 500 replicates
Discussion
As mentioned previously, C. (Hovorkonema) nematodes detected in respiratory tracts of raptors have been identified usually as C. (Hovorkonema) americanaChapin, 1925; C (Hovorkonema) brodskiiSultanov, 1946 and C. (Hovorkonema) variegatum (Creplin, 1849). C. (Hovorkonema) americana was described by Chapin (1925), based on material collected from a host determined as the red-tailed hawk Buteo borealis (actually synonym of Buteo jamaicensis (Gmelin, 1788)) in Virginia, USA. Since description, this species has been detected in a wide range of birds of prey and owls and are considered by several authors (e.g., Borgsteede & Okulewicz, 2001; Borgsteede et al., 2003; Sitko & Okulewicz, 2010) as a rare, typical parasite of respiratory tracts of raptors in the Holarctic Region. However, Ryzhikov (1980) on the basis of experimental infection contested the validity of C. (Hovorkonema) americana and C. (Hovorkonema) brodskii and regarded both as synonym of C. (Hovorkonema) bronchialis (Mühling, 1884) widely recognized as typical parasite of Anseriformes. These conclusions were questioned by Borgsteede and Okulewicz (2001), who on the basis of morphological data and a critical review of existing literature data consider C. (Hovorkonema) americana as a valid taxa. Our results confirmed these statements: clearly demonstrating the existence of the same species of C. (Hovorkonema) in air sacs in birds of prey in Europe and North America – C. (Hovorkonema) americana. Obtained sequences clearly showed almost 100 % homogeneity among nematodes sampled from birds of prey and owls from the Czech Republic, the snowy owl from North America and, in our opinion, sequences obtained by Krone et al. (2007) and identified as “H. variegatum”, clearly consist of two molecularly distinct species and should be treated with considerable caution. Indisputably, as we mentioned previously, some of these represent C. (Hovorkonema) americana. But what about the others? C. (Hovorkonema) variegatum was described by Nathusius (1837) based on material collected from the black stork Ciconia nigra in Germany under the name Strongylus trachealis. Over a period of time, C. (Hovorkonema) variegatum was recognized as a typical parasite of storks and cranes (Vogel, 1928; Ryzhikov, 1949; Okulewicz, 1984), and also reported from the Indian peafowl, Pavo cristatus, in India (Ali, 1970). Moreover, this species was reported frequently in several species of birds of prey in Germany (Lierz et al., 1998; Krone, 2000; Krone et al., 2002; Lierz et al., 2002; Krone et al., 2007), Finland (Krone et al., 2006) and Spain (Sanmartín et al., 2004), but most of the cited authors do not give any morphological and morphometric data about these findings. According to detailed data, included only in the paper of Krone et al. (2007)Cyathostoma nematodes collected from Ciconiform, Falconiform and Gruiform birds were identified according to Hartwich (1994). However, Hartwich (1994), based on existing literature data, synonimised three species of C. (Hovorkonema): the author agreed with the hypothesis formulated by Ryzhikov (1980) and treated C. (Hovorkonema) americana as synonym of C. (Hovorkonema) bronchialis. Furthermore, according to Vogel (1928) and Ali (1970), Hartwich also regarded C. (Hovorkonema) bronchialis as a synonym of C. (Hovorkonema) variegatum. Thus, C. (Hovorkonema) variegatum sensu Hartwich (1994) consisted of three species: C. (Hovorkonema) americana, C. (Hovorkonema) bronchialis and C. (Hovorkonema) variegatum sensu stricto. In light of this, high level of morphometric variability given by Hartwich (1994) as key features for C. (Hovorkonema) variegatum, especially the spiculae length (range 0.320 – 0.870 mm, Table 1) are not reliable. In several species of Cyathostoma spiculae length tends to be a constant feature, characterized by relatively low variability (Kanarek, 2009), and seems to be a good differentiating feature with key significance. According to available literature data (see Table 1), morphology of C. (Hovorkonema) americana and C. (Hovorkonema) variegatum are closely related, but not identical; despite almost identical structure of copulatory bursa (for details and comparison see e.g., Chapin, 1925; Vogel, 1928; Ali, 1970; Okulewicz, 1984; Borgsteede & Okulewicz, 2001) C. (Hovorkonema) variegatum also possess distinctly longer spiculae (0.58 – 0.77 mm) than C. (Hovorkonema) americana (0.47 – 0.511 mm) (Table 1). Other observed morphometric differences, such as dimensions of buccal capsule which is slightly larger in C. (Hovorkonema) variegatum (Table 1) are, in our opinion, not conclusive. What is important, nematodes identified as “H. variegatum”Krone et al. (2007) recorded not only in birds of prey (Accipiter gentilis, A. nissus, Buteo buteo, Circus aeruginosus, Haliaeethus albicilla), but also in common crane Grus grus, typical host of C. (Hovorkonema) variegatum sensu stricto. Unfortunately, sequences of ITS2 gene deposited by Krone et al. (2007) were not adequately described (lacking the name of the hosts), similar to the presented morphological data (lacking such basic facts as name of the hosts and range of the measurements). However, during preparation of the reviewed version of this manuscript the sequence data of Krone et al. (2007) have been updated (last modification 9th of September 2015). Thus, in the light of presented facts, we are convinced, that some sequences deposited in Gen-Bank as “H. variegatum” by Krone et al. (2007) are clearly distinct from sequences obtained in the present work of C. (Hovorkonema) americana and represent sequences of C. (Hovorkonema) variegatum sensu stricto (DQ679967 and AY702698). In our opinion C. (Hovorkonema) americana (typical parasites of tracheae and air sacs of raptors) and C. (Hovorkonema) variegatum (typical parasites of tracheae of cranes and storks) are valid, molecularly and morphologically distinct species. Although, we cannot exclude the possibility that C. (Hovorkonema) variegatum can accidentally parasitize birds of prey and owls (e.g. DQ679967 – Accipiter nisus and AY702698 – Buteo buteo) but this results do not confirm that this phenomenon is common and this issue requires further study. On the other hand, literature data clearly showed, that C. (Hovorkonema) variegatum in typical hosts, such as storks and cranes occurs exclusively in trachea (Vogel, 1928; Okulewicz, 1984), while C. (Hovorkonema) americana in raptors were recorded mainly in air sacs, less frequently in tracheae (Hunter et al., 1993; Borgsteede & Okulewicz, 2001; Krone et al., 2007), but this issue regarding further, detailed analysis.
Comparison of selected biometrical and morphological characteristics of species of C.(Hovorkonema) nematodes recorded in birds of prey: all measurements in millimeters
Accipiter gentilis, A. nisus, B. buteo, C. aeruginosus, Haliaeetus albicilla
Number of teeth
6 – 7
7
7
6 – 7
6 – 7
Head collar
absent
absent
absent
absent
Male: Body length
12
10.815 – 13.420
13.26 – 13.30
7.1 – 9.8 (8.7)
8.11 – 8.43
4 – 14
12.9
Body width
0.487 – 0.545
0.501
0.31 – 0.35
0.25 – 0.6
0.409
Oesophagus length
0.73
0.721 – 0.745
0.714
0.51 – 0.58
0.728
Buccal capsule (width x depth)
0.185 x 0.185
0.175 – 0.187 x 0.165 – 0.174
0.232 x 0.276 – 0.280
0.105 – 0.125 (0.114) x 0.095 – 0.11 (0.106)
0.101 – 0.105 x 0.095 – 0.10 0
.065 – 0.170 x 0.055 – 0.166
0.201 x 0.184
Spicules length
0.47 – 0.49
0.49 – 0.511
0.512
0.58 – 0.77 (0.63)
0.64 – 0.67
in original text 0.64 – 6.67 – probably typographic error surprisingly, with only part of the sequences deposited by Krone et al. (2007) and described as “Hovorkonema variegatum” (Fig. 1).
0.320 – 0.870
0.522
Gubernaculum
present, 0.067 long
present, 0.070 – 0.074 long
absent
present
?
?
?
Female: Body length
up to 30
26.7 – 28.95
18.5 – 19
15.3 – 30.5
25.95
4.9 – 46
29.3
Body width
0.975 – 1.4
1.02
0.68
0.55 – 1.0
1.04
Oesophagus length
0.96
0.908 – 0.952
1.09
0.55 – 1.25 0
.935
Buccal capsule (width x depth)
0.37 x 0.28
0.36 – 0.37 x 0.275 – 0.283
0.408 – 0.544
0.393 – 0.275
0.31 x 0.25
0.11 – 0.56 x 0.08 – 0.368
0.525 x 0.371
Vulvar distance from anterior end
“…vulva just before to the middle of the body…”
13.07 – 14.375
before middle of the body
36 – 40.5 (38.2) % of the body length
10.22
14 – 16
16.94
Tail length
0.350 – 0.377
0.360
0.19 – 0.25
0.22
0.16 – 0.45
0.467
Eggs
0.072 x 0.042
0.074 – 0.076 x 0.042 – 0.044
0.092 – 0.096 x 0.048
0.070 – 0.088 x 0.046 – 0.055
0.072 – 0.080 x 0.049 – 0.051 0
.082 x 0.043
Another interesting issue is the validity of C. (Hovorkonema) bronchialis, a species reported mainly in geese, but also detected in Casuariiformes. Some authors (Ryžikov & Zavadil, 1958; Ali, 1970; Baruš et al., 1978) recognized C. (Hovorkonema) boularti (Megnin, 1884) and C. (Hovorkonema) bronchialis as synonym of C. (Hovorkonema) variegatum. However, Borgsteede and Okulewicz (2001) recognized C. (Hovorkonema) bronchialis as a valid species. Based on literature data (e.g., Griffiths et al., 1954; Ryžikov & Zavadil, 1958; Hernandez-Rodriguez et al., 1975) the structure of copulatory bursa, morphology and morphometry of C. (Hovorkonema) bronchialis, especially spiculae length are closely related to C. (Hovorkonema) variegatum (for details and comparison see e.g., Chapin, 1925; Vogel, 1928; Ryžikov & Zavadil, 1958 and Tables 1 and 2). However, spiculae length provided by Chapin (1925) for C. (Hovorkonema) bronchialis (0.51 – 0.62 mm), differ slightly from measurements provided by Griffiths et al. (1954) (0.54 – 0.70 mm), Ryžikov and Zavadil (1958) (0.58 – 0.72 mm) and Hernandez-Rodriguez et al. (1975) (0.56 – 0.70 mm) (Table 2). Based on this, it is quite possible that C. (Hovorkonema) bronchialis is an synonym of C. (Hovorkonema) variegatum, but this issue requires further research based on detailed molecular and morphological analysis of material collected from typical hosts (Anseriformes).
The last species of C. (Hovorkonema) recorded in birds of prey, C. (Hovorkonema) brodskii was described on the basis of nematodes collected from the Western marsh-harrier Circus aeruginosus, sampled near Tashkent, Uzbekistan, Central Asia (Sultanov, 1946) and has not been recorded since. Therefore its validity is still doubtful. In fact, C. (Hovorkonema) brodskii and C. (Hovorkonema) americana are almost identical, except slightly smaller body dimensions, larger eggs and buccal capsule in C. (Hovorkonema) brodskii (Table 1), but in our opinion these differences are not significant. What is important, speculate length in C. (Hovorkonema) brodskii and C. (Hovorkonema) americana are the same; the only one, well-articulated differentiating feature is absence of gubernaculum in C. (Hovorkonema) brodskii (Table 1). Among Strongyloidea, absence/presence of gubernaculum is recognized as an important feature with significant taxonomic implications e.g., Ali (1970) based his arrangement of subfamily Cyathostominae on the presence/absence of gubernaculum. However, this structure (when it occurs) in Cyathostoma is small, delicate and very easy to overlook, especially in well-cleared material. In fact, it is difficult to resolve whether differences in absence/presence of gubernaculum in morphologically close related species of C. (Hovorkonema) are key differentiating features, or whether they result from variability among specimens or misapprehension in the original descriptions. On the other hand, two C. (Cyathostoma) species occurring in respiratory tract of fish-eating avian hosts, C. (Cyathostoma) phenisci Baudet, 1937 and C. (Cyathostoma) verrucosumHovorka & Macko, 1959 are almost identical, with the exception of the presence of gubernaculum in C. (Cyathostoma) verrucosum; however, molecular analysis showed the identical nature of both species (Kanarek et al., 2013). On this basis, we tend to recognize C. (Hovorkonema) brodskii as a synonym of C. (Hovorkonema) americana.
Comparison of selected biometrical and morphological characteristics of C.(Hovorkonema) bronchialis (Mühling, 1884); all measurements in millimeters
“…posterior part of the anterior third of the body…”
12 – 14.5 mm from anterior end
in first 1/3 length of the body
Tail length
0.16 – 0.30
0.075
0.272 – 0.336
0.230 – 0.263
Eggs
0.074 – 0.083 x 0.049 – 0.062
0.068 – 0.085 x 0.043 – 0.058
0.073 – 0.083 x 0.052 – 0.056
0.082 – 0.086 x 0.059 – 0.065
The present work shows that in the tracheae, bronchii and air sacs in analyzed birds of prey and owls from Europe and North America, only one species of C. (Hovorkonema) occurs, C. (Hovorkonema) americana. However, incidental occurrence of other C. (Hovorkonema) species, typical for other groups of avian hosts, is possible. The result obtained also emphasizes the necessity for careful and critical interpretation of the existing data, regarding morphology and systematics of both subgenera among Cyathostoma. Moreover, this demonstrates the need to use both morphological and molecular methods in the contemporary ecology and systematics of syngamid nematodes.
