Зоологический журнал, 2020, T. 99, № 3, стр. 261-274

Specimen collection

Trematodes were collected from six Kuhl’s pipistrelles specimens; five captured on the 18th September, 2014 and one on the 29th May, 2018 in the Kalinino village (46°20′ N, 48°52′ E), Astrakhan Oblast, Russia (Lower Volga region). Adult worms were collected for morphological study and relaxed in freshwater at room temperature, fixed in 70% ethanol, and subsequently stained with acetocarmine. All measurements were made in micrometers unless otherwise indicated. Mean values are provided in parentheses. Specimens for genetic analysis were fixed in 96% ethanol and stored at +4°C.

Paratype examination

Zdzitowiecki (1969) has described P. oppositus based on holotype and several paratypes. Holotype deposit location remains unknown (according to personal communication by Dr. R. Salamatin). A set of horizontal optical sections of body region between the intestinal bifurcation and the testes of P. oppositus paratype were studied from photographs. The paratype is deposited in the Natural History Museum, London (Parasites & Vectors Division, Department of Life Sciences, cat. # 1992.12.3.25) (Natural History Museum, 2014). Photos were kindly provided by Dr. Rodney Bray.

DNA extraction, amplification, sequencing, and phylogenetic analysis

To obtain LSU sequences, DNA was isolated from one G. oppositus specimen and one Lecithodendrium cf. skrjabini specimen using Chelex-100 and Proteinase K. The D1-D3 regions of 28S rDNA (up to 1,200 bp) were amplified with primer pairs 28Sy/28Sz (Hillis, Dixon, 1991) for Lecithodendrium cf. skrjabini and LSU5/1500R (Tkach et al., 2003) for G. oppositus. Amplicons were purified using a Cleanup mini Purification Kit (Eurogene) and sequenced on the ABI-Prism 3500xl using the same primers.

Newly generated sequences were deposited in GenBank (www.ncbi.nlm.nih.gov) as follows: G. oppositus – MK575195; Lecithodendrium cf. skrjabini – MK575196. The sequences were mounted in general alignment with 61 species (Tkach et al., 2000, 2001, 2003; Olson et al., 2003; Al-Kandari et al., 2011; Galaktionov et al., 2012; Lord et al., 2012; Heneberg, Literák, 2013; Boyce et al., 2014; Presswell et al., 2014; Kanarek et al., 2014, 2015, 2017; Kudlai et al., 2015, 2016; Hernandez-Orts et al., 2016; Bell et al., 2018; Galaktionov, Blasco-Costa, 2018) (Table 1). First, sequences were automatically aligned using the Muscle algorithm (Edgar, 2004) as implemented in SeaView 4.0 (Gouy et al., 2010), and then the alignment was adjusted manually. The final length of alignment was 1267 bp. The phylogenetic analysis was performed using the Maximum Likelihood method as implemented in the PhyML program (Guindon, Gascuel, 2003), with the GTR + G + I model, as suggested by the Modeltest program (Posada, Crandall, 1998). The stability of clades was assessed using a non-parametric bootstrap with 1,000 pseudoreplicates. Bayesian analysis was performed using MrBayes 3.2.6 at the CIPRES Science Gateway (Miller et al., 2010). Trees were run as two separate chains (default heating parameters) for 15 million generations, by which time they had ceased converging: the final average standard deviation of the split frequencies was less than 0.01. The quality of chains was estimated using built-in MrBayes tools and using the Tracer 1.6 package (Rambaut et al., 2014). Based on the estimates by Tracer, the first 7,000 generations were discarded for burn-in.

Systematics

Family Pleurogenidae Looss 1899

Genus Gyrabascus Macy 1935

Gyrabascus oppositus (Zdzitowiecki 1969) comb. n. (fig. 1)

Fig. 1.

Gyrabascus oppositus: A – whole specimen, ventral view; B – ovarian complex, dorsal view; C – terminal genitalia, ventral view; fc – fertilization chamber, ga – common genital atrium, lc – Laurer’s, canal, m – metraterm, ot – ootype with Mehlis’ gland, ov – ovary, pc – prostatic cells, pp – pars prostatica, s – sphincter, sr – seminal receptacle, sv – seminal vesicle, vr – vitelline reservoir. Scale bars (mm): A – 0.4; B, C – 0.05.

Syns: Parabascus oppositus Zdzitowiecki 1969, Parabascus semisquamosum (Braun 1900) sensu Mituch 1965, Parabascus lepidotus Looss 1907 sensu Khotenovsky 1985 pro parte.

Host: Pipistrellus kuhlii (Kuhl 1817) (Chiroptera: Vespertilionidae).

Locality: Kalinino village, Astrakhan Oblast of Russia (46°20′ N, 48°52′ E).

Date of collection: 18 September, 2014 and 29 May, 2018.

Site of infection: intestine.

Prevalence and intensity: in 5 of 6, 5–10 ind.

Specimens deposited: mounts № 14277, 14278 in the Museum of Helminthological Collections at the Centre of Parasitology of the A.N. Severtsov Institute of Ecology and Evolution (IPEE RAS) in Moscow, Russia.

Description (based on 15 adult specimens). Body pyriform with conical anterior extremity and rounded posterior end; length 0.62–1.01 (0.80) mm, maximum width 0.33–0.44 (0.40) mm at level of testes, rare of ventral sucker. Body length to width ratio 1.44–2.57 : 1 (2.02 : 1); body width to length ratio 1 : 0.39–0.69 (1 : 0.51). Forebody 290–491 (390) which represents 38.9–69.4 (50.7)% of body length. Tegument spinose. Oral sucker drop-shaped with elongated antero-dorsal lobe, 89–108 (97) × 57–70 (64). Mouth opening subterminal. Prepharynx usually not observed. Pharynx 38–51 (43) × 38–51 (45). Oral sucker to pharynx width ratio 1.25–1.67 : 1 (1.44 : 1). Oesophagus 120–215 (163); intestinal bifurcation in posterior third of forebody. Intestinal branches ending blindly far behind testes. Ventral sucker transverse-oval and recessed into body, 82–108 (95) × 108–133 (123). Oral sucker to ventral sucker width ratio 0.50–0.55 : 1 (0.53 : 1). Two testes, round to oval, entire, opposite, in anterior half of hindbody; sinistral 95–152 (122) ×  76–138 (122), dextral 90–139 (125) × × 76–138 (110). Cirrus sac absent. Seminal vesicle tubular, convoluted, free in parenchyma. Pars prostatica tubular, surrounded by extensive field of prostatic cells. Ejaculatory duct short, opening into tubular common genital atrium. Genital pore submedian, at level of ventral sucker, on opposite side to ovary. Ovary round to oval, entire, sinistral or dextral, at level of ventral sucker; 82–114 (98) × 95–152 (115). Proximal part of oviduct forms vesicular fertilization chamber. Seminal receptacle canalicular. Laurer’s canal opens on dorsal side of body in ventral sucker region. Ootype with Mehlis’ gland and vitelline reservoir median, between ventral sucker and testes. Vitellarium follicular in posterior half of forebody but overlaps with ventral sucker to some extent; follicles in two sublateral fields confluent dorsally. Uterus with transversal loops fills almost all space in hindbody. Metraterm with somewhat thickened walls, terminating with sphincter, and opening into common genital atrium ventral to male pore. Eggs numerous, oval, operculate, 24–26 (26) × × 13–16 (13). Excretory pore terminal; vesicle I-shaped and usually reaches posterior edge of testes.

Remarks. Our specimens are consistent with the prominent morphological features of P. oppositus, as described by Zdzitowiecki (1969), specifically the body size and shape, configuration of the inner organs, ratio of the suckers, and eggs size. In Zdzitowiecki’s (1969) paper, no data exists on morphology of the male terminal genitalia, however drawing of P. oppositus made by Mituch (1965) (named as P. semisquamosum by this author) does not indicate that the seminal vesicle is enclosed into the cirrus-sac.

Only the tubular pars prostatica with a field of prostate cells were visible on photos of the paratype terminal genitalia, while the wall of the cirris-sac separating this part of the male duct from the parenchyma is absent. Thus, the paratype of P. opposites obviously does not have a cirrus-sac.

Family Lecithodendriidae Looss 1902

Genus Lecithodendrium Lühe 1896

Lecithodendrium cf. skjabini Mazaberidse 1963 (fig. 2)

Fig. 2.

Lecithodendrium cf. skrjabini, whole view. Scale bar 0.2 mm.

Host: Pipistrellus kuhlii (Kuhl 1817) (Chiroptera: Vespertilionidae).

Locality: Kalinino village, Astrakhan Oblast of Russia (46°20′ N, 48°52′ E).

Date of collection: 29 May, 2018.

Site of infection: intestine.

Prevalence and intensity: in 1 of 6, 22 ind.

Specimens deposited: mounts № 14 279, 14 280 in the Museum of Helminthological Collections at the Centre of Parasitology of the A.N. Severtsov Institute of Ecology and Evolution (IPEE RAS), in Moscow, Russia.

Description (based on 11 adult specimens). Body fusiform; length 1.04–1.33 (1.18) mm, maximum width 0.30–0.40 (0.38) mm at mid-level of body. Body length to width ratio 2.56–3.61 : 1 (3.17 : 1); body width to length ratio 1 : 0.28–0.39 (1 : 0.32). Forebody 390–516 (440), which represents 32.3–40.2.4 (37.4)% of body length. Tegument unarmed. Oral sucker rounded, 60–76 (67) × 57–79 ( 68). Mouth opening subterminal. Prepharynx absent, pharynx 19–28 (23) ×  25–32 (27). Oral sucker to pharynx width ratio 2.00–3.00 : 1 (2.60 : 1). Oesophagus 222–294 (251), intestinal bifurcation in forebody. Intestinal branches end near anterior edge of testes. Ventral sucker spherical to subsperical, 63–82 (75) × 63–82 (70). Oral sucker to ventral sucker width ratio 0.77–1.10 : 1 (0.98 : 1). Testes two, entire, round to oval, opposite, at level of ventral sucker; sinistral 82–127 (114) × 82–120 (104), dextral 95–133 (124) × 95–133 (110). Pseudocirrus-sac elongate-oval, 125 × 62–76 (70), with convoluted seminal vesicle, lies transversely to ventral surface of body and communicates with short thick-walled common genital atrium. Genital pore median or slightly sinistro-submedian, near anterior edge of ventral sucker. Ovary elongate-oval, entire, median, or slightly sinistro-median or dextro-submedian; post-testicular; contiguous with testes or separated, 95–133 (113) × 76–120 (97). Seminal receptacle canalicular. Laurer’s canal opens on dorsal side of body, at level of posterior edge of ovary or distinctly posterior to ovary. Ootype with Mehlis’ gland and vitelline reservoir near postero-sinistral margin of ovary or at level of posterior half ovary. Vitellarium follicular; elongated follicles in two dorsal clusters, 7–9 in each. Clusters postovarian but partly overlapped by ovarian area. Uterus fills almost all space in hindbody. Metraterm of similar length to pseudocirrus-sac, running over posterior surface of pseudocirrus-sac, and opening into common genital atrium. Eggs numerous, oval, operculate, 19–22 (20) × 10–13 (10). Excretory pore terminal; vesicle V-shaped and reaches to testes.

Remarks. Previous descriptions of L. skrjabini were deficient. Matsaberidze (1963) and Matsaberidze and Khotenovsky (1967) presented a description based on deformed specimens with compressed forebodies (Matsaberidze, 1963; Matsaberidze, Khotenovsky 1967). Kirillov et al. (2012) described specimens with a stretched forebody, as indicted by the equatorial position of the ventral sucker. Also, in the paper of Matsaberidze and Khotenovsky (1967) there is confusion with the drawing of L. skrjabini, which is replaced with the drawing of L. linstowi Dollfus 1931. The actual drawing of L. skrjabini is depicted in figure 1.

Comparison of the descriptions published by Matsaberidze (1963), Matsaberidze and Khotenovsky (1967) and Kirillov et al. (2012) enable the reconstruction of the body shape of relaxed L. skrjabini adults: it is fusiform, while the ventral sucker occupies the pre-equatorial position. The samples within this study are similar to L. skrjabini in terms of body shape, internal organ position, sucker size and ratio, the vitellarium morphology, and egg size. However, these trematodes differ from L. skrjabini in terms of body length and forebody and oesophagus lenght. This study presents specimens lengths of 1.04–1.33 mm, and the maximum length of L. skrjabini is 0.89 mm (Matsaberidze, Khotenovsky 1967). However, the publications of Matsaberidze (1963), and Matsaberidze and Khotenovsky (1967) take measurements from worms with compressed forebodies. At the same time, the adults in this study are evidently larger than the individuals collected by Kirillov et al. (2012). It is put forward that this difference is due to the phenomenon of host-induced morphological variability.

Molecular phylogenetic analysis

In both the ML and BI analysis, G. oppositus appears as a member of the well-supported Gyrabascus spp. clade that is also includes G. amphoraeformis (Mödlinger 1930) and Gyrabascus sp. (fig. 3). However, the phylogenetic relationships among representatives within the Gyrabascus spp. clade are poorly resolved. In turn, the Gyrabascus spp. clade is situated within the major Pleurogenidae + Collyriclidae clade. The Pleurogenidae + Collyriclidae clade appears to be monophyletic, with statistically significant support. Within the Pleurogenidae + Collyriclidae clade, the Gyrabascus spp. appears a sister clade to the group, containing representatives of the genera Cortrema Tang 1951, Leyogonimus Ginetsinskaya 1948 and Macyella Neiland 1951, but support of this sister connection is weak.

Fig. 3.

Phylogenetic position of Gyrabascus oppositus and Lecithodendrium cf skrjanbini based on the analysis of 28S rDNA partial sequences using ML and BI algorithms. Nodal support: ML/BI. The clade which is absent in ML, but present in BI noted with asterisk. Newly obtained sequences are shown in bold. Plagiorchis elegans is designated as outgroup taxa.

Lecithodendrium cf. skrjabini together with congeneric species form the Lecithodendrium spp. clade, which is strongly supported by BI analysis and moderately supported by ML analysis. Inside this clade, Lecithodendrium cf. skrjabini appears as a weakly supported sister taxon to L. linstowi. The Lecithodendrium cf. skrjabini + L. linstowi group is combined with the Lecithodendrium sp. into a well-supported monophyletic clade. In turn, the Lecithodendrium cf. skrjabini + + L. linstowi + Lecithodendrium sp. clade is strongly supported by BI analysis and moderately supported by ML analysis sister group to Lecithodendrium spathulatum (Ozaki 1929). The Lecithodendrium spp. clade appears as a member of the family Lecithodendriidae (fig. 3). The family Lecithodendriidae is closely related to Stomylotrematidae, however, this is weakly supported in both ML and BI analyses. The Lecithodendriidae + + Stomylotrematidae group is clustered with the Phaneropsolidae, strongly supported by BI analysis and moderately supported by ML analysis. The Lecithodendriidae + Stomylotrematidae + Phaneropsolidae clade is a sister group to the family Microphallidae, this is also strongly supported by BI analysis and moderately supported by ML analysis.

Meanwhile, the major clade of trematodes containing the Lecithodendriidae, Stomylotrematidae, Phaneropsolidae and Microphallidae appears as a low-supported sister group to the Pleurogenidae + + Collyriclidae clade (fig. 3). In turn, the [(Lecithodendriidae +Stomylotrematidae + Phaneropsolidae) + + Microphallidae] + (Pleurogenidae + Collyriclidae) clade is nested within the well-supported monophyletic group of microphalloids, that also includes the Prosthogonimidae. The Pachypsolidae occupies a basal position to all of the aforementioned taxa.

Genus Gyrabascus Macy 1935 emend

Diagnosis (based on Lotz, Font (2008) with changes). Body broadly oval, nearly as wide as long. Oral sucker terminal to subterminal. Ventral sucker equatorial or preequatorial. Prepharynx very short. Testes in middle third of body. Cirrus-sac absent. Seminal vesicle lies transversely or irregularly coiled. Genital pore marginal or submedian, sinistral or dextral, locates in region of ventral sucker, opposite to ovary, not anterior to vitellarium. Ovary entire to lobed, extends anterior to testes. Uterus comprised of overlapping coils that fill posterior half of body. Eggs small, numerous. Vitellarium follicular, in forebody, sometimes reaches into ventral sucker region. In bats and rodents; North America and Eurasia. Type species G. brevigastus Macy 1935.

Integration of the Gyrabascus spp. into the family Pleurogenidae (fig. 3) can be verified through phylogenetic reconstructions from other authors (Tkach et al., 2002, 2003; Kanarek et al., 2014, 2015, 2017; Bell et al., 2018). However, in phylogram, presented in this article, the family appears to be paraphyletic, because Langeronia macrocirra Caballero et Bravo 1949 (Pleurogenidae) forms a highly supported sister relationship with Collyriclum faba (Bremser in Schmalz 1831) (Collyriclidae). Earlier, the sister relationship between C. faba and L. macrocirra was demonstrated by Heneberg and Literák (2013). The combination of C. faba with pleurogenid trematodes is supported by the morphological data on cercariae. Cercaria of C. faba, has four pairs of penetration glands and a bilobed virgula in the posterior half of the oral sucker, similar to the larvae of other pleurogenids (Heneberg et al., 2015).

The results of Kanarek et al. (2014, 2015, 2017) and Bell et al. (2018) indicate a close relationship between the genera Gyrabascus and Cortrema. According to our own data, there is no direct phylogenetic relationship between these taxa, as the only species of the genus Cortrema that has been sequenced, Cortrema magnicaudata (Bykhovskaya-Pavlovskaya 1950), is clustered with two members of the genus Macyella: Macyella massanae (Vaucher 1968) and Macyella postgonoporus Neiland 1951. Furthermore, M. massanae was closer to C. magnicaudata than to congeneric species (fig. 3). It should be noted that previous to reassigning M. massanae to the genus Macyella, based on the phylogenetic data of Kanarek et al. (2017), it was considered to be member of the genus Collyricloides Vaucher 1968. Consequently, a sister group to the Gyrabascus spp. clade cannot be clearly supported based on our own phylogenetic data.

The presented molecular phylogenetic data clearly demonstrates the monophyly of the genus Lecithodendrium and the family Lecithodendriidae as a whole. The data also clearly indicate the close relationships Lecithodendriidae with the families Phaneropsolidae and Stomylotrematidae, which supports the results of a recent study by Bell et al. (2018). In the study by Bell et al. (2018), the relationship of the Phaneropsolidae + + Stomylotrematidae + Lecithodendriidae clade with the Pleurogenidae, Prosthogonimidae, and Microphallidae was unclear. According to the results of our phylogenetic analysis, the Phaneropsolidae + Stomylotrematidae + Lecithodendriidae clade has a common ancestor with microphallid trematodes. However, the relationships between the Prostogonimidae and the Pleurogenidae + Collyriclidae and Microphallidae + + (Phaneropsolidae + Stomylotrematidae + Lecithodendriidae) clades were poorly resolved.