A new filarial nematode, Litomosa vetuni Pikula, Bednarikova & Řehulková n. sp. (Nematoda: Onchocercidae), from the parti-coloured bat Vespertilio murinus in Europe: An integrative species description

Abstract

A new filarial nematode, Litomosa vetuni n. sp. (Onchocercidae), is described from the parti-coloured bat, Vespertilio murinus, in the Czech Republic. Adult worms were recovered from the abdominal, thoracic and uterine cavities. The new species is the fourth species of Litomosa reported from this host and the first characterised using an integrative approach combining morphology, molecular data (cox1 and 12S rDNA) and scanning electron microscopy (SEM). SEM observations provide the first available surface images for a species of Litomosa, showing the position of the amphids and prominent cephalic papillae at the anterior extremity, as well as the arrangement of three caudal lappets in both sexes. Litomosa vetuni n. sp. is distinguished from morphologically similar congeners by the shape and proportions of the segmented buccal capsule, the morphology of the spicules, and the absence of an area rugosa. Both cox1 and 12S rDNA sequences from the holotype, paratype and their microfilariae formed a well-supported, internally consistent lineage within Litomosa. However, due to the extremely limited availability of reference sequences and the lack of morphological vouchers for other published Litomosa haplotypes, broader phylogenetic placement of the new species within the genus remains unresolved. The study highlights inconsistencies in published morphological descriptions and the scarcity of molecular data for Litomosa spp., emphasising the need for a comprehensive taxonomic revision of bat-associated filarial nematodes using integrative approaches.

Graphical abstract

Highlights

• •A new cavity-dwelling filarial nematode, Litomosa vetuni n. sp., described from Vespertilio murinus in the Czech Republic.
• •This is the first description of a species of Litomosa using light and scanning electron microscopy.
• •Phylogenetic analyses based on cox1 and 12S rDNA sequences confirmed the distinct species status of L. vetuni n. sp.
• •The taxonomy of bat-associated filarial parasites needs an integrative revision.

1. Introduction

The parti-coloured bat (Vespertilio murinus Linnaeus, 1758) is a temperate Eurasian bat species with a distribution range spanning from eastern France across Europe, bounded by southern Scandinavia and the northern Balkan Peninsula, and extending eastward to China, northern Pakistan and the Korean Peninsula (Rydell and Baagøe, 1994; Shpak, 2017). It inhabits a wide range of landscapes, from forests and urban areas to treeless steppes and agricultural regions (Rydell and Baagøe, 1994). While insects emerging over water, along with small dipterans up to 10 mm in length and other air-borne prey (e.g. aphids), constitute its main food sources throughout the year, the species feeds primarily on beetles and moths near streetlamps in late summer and autumn (Bauerová and Ruprecht, 1989; Rydell and Baagøe, 1994). Insects are caught by aerial hawking only.

Vespertilio murinus is known to host three species of filarial nematodes assigned to Litomosa Yorke & Maplestone, 1926 (Nematoda: Onchocercidae) (see Table 1). Members of this genus parasitize numerous bat families, including the Emballonuridae, Hipposideridae, Megadermatidae, Miniopteridae, Molossidae, Pteropodidae, Rhinolophidae and Vespertilionidae, with most species reported from the Old World. In addition, two species have been recorded from rodents of the Geomyidae in the Nearctic Region (Gardner and Schmidt, 1986). Species of Litomosa have been documented across a broad geographical range, including the Afrotropical Region (Africa and Madagascar), the Palearctic Region (Europe, Japan and parts of western and central Asia, including Turkey, Azerbaijan, Georgia and Uzbekistan), the Oriental Region (India and Malaysia), the Australasian Region (Australia and the Bismarck Archipelago) and the Nearctic Region (North America). A total of 23 Litomosa species are currently recognised based on published descriptions and available taxonomic names (see Table 1 for a summary of the nominal species, their hosts, and localities). Litomosa hanaki Hošek, 1985 is considered herein a nomen nudum, as no formal description or verifiable record exists in the scientific literature, and it is therefore not included in Table 1.

Table 1.

Nominal species of Litomosa with synonyms, hosts, localities, and references. Some data are summarized from Sonin (1975), who also lists the original sources. An asterisk (∗) indicates the type-host/type-locality.

Litomosa species	Synonym	Host species	Host family	Locality	Reference
Litomosa adami Petit, 1980		Miniopterus minor minor∗	Miniopteridae	Africa (DR Congo; Republic of the Congo∗)	Petit (1980)
		Miniopterus natalensis
Litomosa aelleni Tibayrenc, Bain & Ramachandran, 1979		Tadarida teniotis∗	Molossidae	Europe (Switzerland)	Tibayrenc et al. (1979)
Litomosa americana McIntosh, 1932		Nycticeius humeralis	Vespertilionidae	North America (USA)	McIntosh (1932); Sonin (1975)
Litomosa beshkovi Jancev, 1971		Pipistrellus nathusii∗	Vespertilionidae	Europe (Bulgaria)	Jancev (1971); Sonin (1975)
Litomosa chiropterorum Ortlepp, 1932		Laephotis capensis	Vespertilionidae	Africa (DR Congo, South Africa∗); Asia (Turkey)
		Miniopterus natalensis∗	Miniopteridae
		Miniopterus schreibersii
		Plecotus auritus	Vespertilionidae
		Rhinolophus acrotis	Rhinolophidae
		Tadarida teniotis	Molossidae
Litomosa dogieli Bogdanov & Vladimirov, 1956	Litomosa desportesi Bain, 1966	Myotis daubentonii	Vespertilionidae	Asia (Uzbekistan∗); Europe (Bulgaria, France, Hungary, Switzerland)	Bogdanov and Vladimirov (1956); Sonin (1975)
		Myotis emarginatus∗
		Myotis myotis
Litomosa filaria (van Beneden, 1872) Yorke & Maplestone, 1926	Litosoma filaria	Barbastella barbastellus	Vespertilionidae	Asia (Turkey, Uzbekistan); Europe (Belgium∗, Bulgaria, Czech Republic, France, Germany, Hungary, Moldova, Poland, Romania, Spain, Ukraine)	van Beneden (1872); Yorke and Maplestone (1926); Sonin (1975)
		Miniopterus schreibersii	Miniopteridae
		Myotis bechsteini	Vespertilionidae
		Myotis daubentoni
		Myotis emarginatus
		Myotis myotis
		Myotis nattereri
		Plecotus auritus∗
		Plecotus austriacus
		Rhinolophus ferrumequinum	Rhinolophidae
		Vespertilio murinus	Vespertilionidae
Litomosa fotedari (Gupta & Trivedi, 1989) Martin, Bain, Jouvenet, Raharimanga, Robert & Rousset, 2006	Litomosoides fotedari	Taphozous melanopogon	Emballonuridae	Asia (India)	Gupta and Trivedi (1989)
Litomosa goodmani Martin, Bain, Jouvenet, Raharimanga, Robert & Rousset, 2006		Miniopterus gleni	Miniopteridae	Madagascar	Martin et al. (2006)
Litomosa hepatica (von Linstow, 1897) Guerrero & Bain, 2011	Filaria hepatica	Pteropus neohibernicus	Pteropodidae	Bismarck Archipelago, West Pacific	von Linstow (1897)
Litomosa hugoti Petit, 1980		Rhinolophus sylvestris	Rhinolophidae	Africa (Gabon)	Petit (1980)
Litomosa jairajpurii Trivedi & Gupta, 1987		Hipposideros fulvus pallidus	Hypposideridae	Asia (India)	Trivedi and Gupta (1987)
Litomosa japonica Ohbayashi & Kamiya, 1979		Vespertilio sinensis	Vespertilionidae	Asia (Japan)	Ohbayashi and Kamiya (1979)
Litomosa maki Tibayrenc, Bain, Ramachandran, 1979		Pteropus vampyrus	Pteropodidae	Asia (Malaysia)	Tibayrenc et al. (1979)
Litomosa miniopteri Mackerras, 1962		Miniopterus schreibersii	Miniopteridae	Australia	Mackerras (1962)
Litomosa ottavianii Lagrange & Bettini, 1948	Litomosa beaucournui Bain, 1966	Miniopterus schreibersii	Miniopteridae	Europe (Bulgaria, France, Italy∗)	Lagrange and Bettini (1948); Sonin (1975)
		Myotis emarginatus	Vespertilionidae
		Rhinolophus blassi	Rhinolophidae
		Rhinolophus hipposideros
		Vespertilio murinus	Vespertilionidae
Litomosa pujoli Bain, 1966		Doryrhina cyclops	Hypposideridae	Africa (Central African Republic)	Bain (1966)
Litomosa seurati Martin, Bain, Jouvenet, Raharimanga, Robert & Rousset, 2006	Litomosa beaucournui Bain, 1966 (in part)	Rhinolophus ferrumequinum	Rhinolophidae	Africa (Algeria∗, Morocco); Europe (France)	Seurat (1921); Bain (1966)
		Rhinopholus sp.
Litomosa skarbilovitschi Petrov & Tschertkova, 1954		Miniopterus schreibersii	Miniopteridae	Asia (Azerbaijan, Georgia, Uzbekistan∗)	Petrov and Chertkova (1954); Sonin (1975)
		Myotis emarginatus	Vespertilionidae
		Rhinolophus bocharicus∗	Rhinolophidae
		Rhinolophus hipposideros
		Rhinolophus mehelyi
Litomosa tewarii (Gupta & Trivedi, 1989) Martin, Bain, Jouvenet, Raharimanga, Robert & Rousset, 2006	Litomosoides tewarii	Lyroderma lyra	Megadermatidae	Asia (India)	Gupta and Trivedi (1989)
Litomosa thomomydis (Gardner & Schmidt, 1986) Guerrero, Martin, Gardner & Bain, 2002	Litomosoides thomomydis	Thomomys talpoides	Geomyidae	North America (USA - Colorado)	Gardner and Schmidt (1986)
Litomosa vaucheri Petit, 1980		Vespertilio murinus	Vespertilionidae	Europe (Switzerland)	Petit (1980)
Litomosa westi (Gardner & Schmidt, 1986) Guerrero, Martin, Gardner & Bain, 2002	Litomosoides westi	Geomys bursarius	Geomyidae	North America (USA - Colorado)	Gardner and Schmidt (1986)

The life cycle of Litomosa spp. remains poorly understood. Junker et al. (2009) proposed macronyssid mites as possible vectors of species of Litomosoides, a genus considered closely related to Litomosa (Guerrero et al., 2003). Ramasindrazana et al. (2016) later emphasised the need for increased molecular screening of a wide range of bat ectoparasites collected at sites where hosts tested positive for filarial nematodes. This includes bat flies, fleas, mites, ticks and other blood-sucking dipterans (see Léger, 2020 for an overview). Recent molecular studies have indeed detected microfilarial DNA of Litomosa sp. in several ectoparasites of V. murinus (Bednarikova et al., 2025a, 2025b); however, the macronyssid mite Steatonyssus spinosus appears to be the most plausible candidate vector as it has repeatedly tested PCR-positive for Litomosa sp. (Bednarikova et al., 2025a).

At present, sequence data (the mitochondrial 12S rRNA and cytochrome c oxidase subunit 1 (cox1 or COI) genes) for Litomosa spp. available in GenBank is combined with morphological species identification for just two species: Litomosa chiropterorum (Junker et al., 2009) and Litomosa westi (Casiraghi et al., 2004), while other Litomosa spp. sequences available in GenBank mostly represent the partial cox1 gene and lack assignment to morphologically described species. Thus, while recent molecular studies have highlighted the diversity of filarial nematodes in bats (Ramasindrazana et al., 2016; Bednarikova et al., 2025a, 2025b), linking gene sequences with morphological characteristics will improve phylogenetic interpretation (Bain et al., 2008).

The present study builds upon recent molecular surveys of filarial nematodes in Vespertilio murinus (Pikula et al., 2023; Bednarikova et al., 2025a), which revealed a distinct and previously unnamed lineage of Litomosa. Here, we provide a formal morphological description of Litomosa vetuni n. sp. based on adult specimens, link this species to previously generated molecular data, and clarify its phylogenetic relationships within the genus using 12S rDNA and cox1 sequences.

2. Materials and methods

2.1. Specimen collection and necropsy

Between 2010 and 2021, 28 parti-coloured bat cadavers were obtained from synanthropic habitats in the Czech cities of Blížkovice (48°59′59″N, 15°50′5″E), Brno (49°11′43″N, 16°36′30″E), Mělník (50°21′2″N, 14°28′27″E), Mníšek pod Brdy (49°52′0″N, 14°15′43″E) and Prague (50°5′15″N, 14°25′17″E) and examined for parasites (Pikula et al., 2023; Bednarikova et al., 2025a). The cadavers, which were obtained from wildlife rescue centres, were dissected at the University of Veterinary Sciences, Brno, Czech Republic. Adult filarial worms were found in five of the 28 bats examined (males n = 4, females n = 1), either in the abdominal or thoracic (one case) cavities during necropsy, with the number of worms ranging from one to eight per bat. Two worms were retrieved from the uterine cavity of a mid-gestation pregnant female.

2.2. Morphological examination (light microscopy and SEM)

The nematodes recovered were washed in 0.9% physiological saline and immediately fixed in 70% ethanol. For morphological examination by light microscopy, nematodes were cleared in a glycerine: water solution (3:7) and examined under a DIC-equipped light microscope (Olympus BX51). Measurements were taken using a digital image analysis system (analySIS auto 5.0), and line drawings were prepared with the aid of a drawing tube. Following examination, specimens were preserved in vials containing 70% ethanol. Blood smears to reveal circulating microfilariae could not be obtained from the cadavers. For SEM, one male and one female worm were used. Fixed specimens were dehydrated through a graded ethanol series and critical-point dried in a Bal-Tec CPD 030 Critical Point Dryer (BAL-TEC AG, Balzers, Liechtenstein) using liquid CO₂. They were then mounted on aluminium stubs with a double-sided adhesive disc and sputter-coated with gold in a Balzers SCD 040 sputtering device (BAL-TEC AG, Balzers, Liechtenstein). The samples were examined with a MAGELLAN 400 scanning electron microscope (Thermo Fisher/FEI, Eindhoven, the Netherlands) at an accelerating voltage of 5 kV. All measurements in the description/table are in micrometres, unless otherwise indicated.

Type-specimens of the new species were deposited in the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic (IPCAS).

2.3. Molecular and phylogenetic analyses

The DNA extraction and primary molecular identification, based on partial mitochondrial cox1, had been undertaken in previous studies (Pikula et al., 2023; Bednarikova et al., 2025a). For further phylogenetic analysis, a standard PCR amplification targeting a 450-bp fragment of 12S rDNA was performed using the primers 12SF (5′-GTT CCA GAA TAA TCG GCT A-3′) and 12SR (5′-ATT GAC GGA TGR TTT GTA CC-3′) (Casiraghi et al., 2004). The PCR reaction was performed in a total volume of 20 μl comprising 10 μl of Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 mM of each primer, 2 μl of template DNA and PCR-grade water, with negative (PCR-grade water) and positive (DNA isolated from Dirofilaria repens) controls. The reaction was set according to the Master Mix protocol with an annealing temperature of 57 °C. Serva DNA Stain G (Serva Electrophoresis, Heidelberg, Germany) and UV light were used to visualise the PCR products obtained on 1.5% agarose gel. Appropriately sized PCR products were purified using the NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany) and sent for commercial Sanger sequencing (SEQme, Dobříš, Czech Republic). The sequences thus obtained were edited using GeneiousPrime software (Biomatters, Auckland, New Zealand) and compared to sequences available in the GenBank database using the BLAST algorithm.

Based on sequences obtained in this and our previous studies (Pikula et al., 2023; Bednarikova et al., 2025a, 2025b), two phylogenetic analyses based on the available cox1 and 12S rDNA sequences of Litomosa spp., Litomosoides spp., and closely related genera were conducted. Both analyses used representative sequences longer than 200 bp (12S rDNA) or 300 bp (cox1). All phylogenies were inferred using IQ-TREE v. 1.6.12 (Nguyen et al., 2015) and the best-fit evolution model selected based on the Bayesian information criterion, computed and implemented using ModelFinder (Kalyaanamoorthy et al., 2017); these were TIM3+F+G4 (12S alignment) and TIM3+F+R4 (cox1 alignment). Branch support was assessed by ultrafast bootstrap (UFBoot) approximation (Minh et al., 2013) and the Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) (Guindon and Gascuel, 2003). The trees were visualised and edited in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and Inkscape v. 1.3 (https://inkscape.org). Details on used model and alignment are included into Figure legends.

3. Results

3.1. Litomosa vetuni Pikula, Bednarikova & Řehulková n. sp.

3.1.1. Taxonomic summary

Type-host: Vespertilio murinus Linnaeus, 1758 (Vespertilionidae), parti-coloured bat.

Type-locality: Synanthropic habitat in the city of Brno (49°11′43″N, 16°36′30″E), Czech Republic.

Other localities: Synanthropic habitat in the city of Prague (50°5′15″N, 14°25′17″E), Czech Republic (Bednarikova et al., 2025a) and the following natural habitats: Lukashino region of Western Siberia (57°19′N, 64°59′E), Russia (Bednarikova et al., 2025a); Gethashen Cave in Gndevang Canyon (39°41′59.8″N, 45°33′43.8″E), Armenia (Bednarikova et al., 2025b); and Ghliana Cave near the town of Tskaltubo (42°22′24.6″N, 42°35′50.7″E), Georgia (Bednarikova et al., 2025b).

Type-material: The holotype (male, collected on 19 October 2021) and seven paratypes (males and females, collected on 19 October and 10 December 2021) were deposited in the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic (catalogue number: IPCAS N-1293).

Site in host: Body cavities: abdominal, thoracic, uterine.

Prevalence and intensity of infection: 17.86% of bats harboured adult nematodes (5/28); intensity ranged from 1 to 8 adult nematodes per infected bat. Microfilariae were detected in 81.5% of bats by molecular testing of host tissues (Bednarikova et al., 2025a).

Representative DNA sequences: The 12S rDNA sequences from the holotype and paratypes were identical; therefore, a single representative sequence was deposited in GenBank (PV364979). The cox1 sequences are deposited under accession numbers OP796371 (holotype), PQ042406 (paratype), OP796369 (microfilariae from the holotype specimen), and PQ042404 (microfilariae from the paratype specimen). The previously published molecular records comprise accessions OP796363-OP796372, PQ042391-PQ042417, PV023858-PV023875 (see Supplementary Table S1 for details).

ZooBank registration: To comply with the regulations set out in Article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN, 2012), details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:4BA8E2AB-0082-4C78-8756-6DDB3129FE10. The LSID for the new name Litomosa vetuni Pikula, Bednarikova & Řehulková n. sp. is urn:lsid:zoobank.org:act:44194FE4-56BC-4423-899F-84A154061354.

Etymology: The species epithet vetuni is derived from the acronym of the University of Veterinary Sciences Brno (VETUNI), in honour of the institution where the species was discovered. It is treated as an invariable noun in apposition.

3.1.2. Description

General [Table 2, Table 3; Fig. 1, Fig. 2, Fig. 3.]. Body filiform, tapering posteriorly (Fig. 1C and D); cephalic extremity with distinct lateral shoulders (Fig. 2A–E, F); caudal extremity bearing three spine-like lappets in both sexes (Fig. 1C and D). Cuticle whitish to brownish (in fixed specimens), finely striated under SEM (Fig. 2, Fig. 3). Head rounded (Fig. 1A), with low apical areola bearing minute circular mouth (Fig. 2A–C); lips absent (Fig. 2C). Amphids paired, situated bilaterally on areola margin, each on slightly elevated circular area with central shallow depression bearing roughened, hair-like surface (Fig. 2C, F). Pair of cephalic papillae located relatively close together on one side of apical region, nipple-like, with flattened apices (Fig. 2B–F). No additional cephalic papillae were observed in the SEM images; no en face view was obtained. Buccal capsule nearly as long as wide, four-segmented; second segment reduced, third segment triangular and enlarged; buccal cavity bottle-shaped (Fig. 1B). Oesophagus muscular, without glandular region; nerve-ring slightly anterior to mid-length of oesophagus (Fig. 1A). Excretory pore visible as shallow depression near anterior extremity (Fig. 2E).

Table 2.

Comparative morphometric data for females of Litomosa vetuni n. sp., morphologically similar congeners (L. ottavianii and L. seurati), and the closest sequenced species (L. chiropterorum).

Species	Litomosa vetuni n. sp.	Litomosa ottavianii		Litomosa seurati	Litomosa chiropterorum
Host	Vespertilio murinus	Miniopterus schreibersii; Vespertilio murinus	Miniopterus schreibersii	Rhinolophus ferrumequinum	Miniopterus natalensis
Morphological character	Present study	Lagrange and Bettini (1948)	Bain (1966)	Bain (1966)	Junker et al. (2009)
Body length (mm)	24 (20–30) (n = 6)	53	42–57	19–25	72 (n = 1)
Body width	161 (150–167) (n = 5)	222	125–230	110–115	169 (150–180) (n = 3)
Body width at level of vulva	120 (109–125) (n = 4)
Buccal capsule	7–14 × 10–16 (n = 6)		15 –19 × 16–22	14–15 × 14–15	17–18 × 20–27 (n = 3)
Nerve-ring to anterior extremity	229 (185–248) (n = 6)		200–230	140–190
Oesophagus length	520 (457–560) (n = 6)	510	450–580	370–420	506 (480–528) (n = 3)
Oesophagus max. width	35 (28–56) (n = 6)
Distance vulva from apex	643 (493–737) (n = 6)	653	450–550	360–515	707 (500–820) (n = 3)
Tail length	123 (102–150) (n = 6)	177	150–210	95–155	182 (142–220) (n = 3)
Mid-tail width	53 (45–59) (n = 6)
Tail width at level of anus	61 (54–72) (n = 6)				70 (55–80) (n = 3)
Caudal lappets	3 large (n = 5)	2 large + 3–4 small	2 small + 3 small	2 large + 1 small	2 large (n = 3)
Caudal lappets length	10–11/9–11/10–11 (n = 3)
Microfilaria detected	yes (n = 5)	yes			yes (n = 1)

Abbreviation: n, number of specimens measured.

Table 3.

Comparative morphometric data for males of Litomosa vetuni n. sp., morphologically similar congeners (L. ottavianii and L. seurati), and the closest sequenced species (L. chiropterorum).

Species	Litomosa vetuni n. sp.	Litomosa ottavianii		Litomosa seurati	Litomosa chiropterorum
Host	Vespertilio murinus	Miniopterus schreibersii; Vespertilio murinus	Miniopterus schreibersii	Rhinolophus ferrumequinum	Miniopterus natalensis
Morphological character	Present study	Lagrange and Bettini (1948)	Bain (1966)	Bain (1966)	Junker et al. (2009)
Body length (mm)	13 (12–14) (n = 3)	27			35 (28–41) (n = 5)
Body width	93 (85–100) (n = 3)	144			105 (90–120) (n = 5)
Buccal capsule	7–9 × 13–14 (n = 2)				13–16 × 17–22 (n = 5)
Nerve-ring to anterior extremity	202 (199–205) (n = 2)
Oesophagus length	377 (288–468) (n = 3)	428			480 (440–550) (n = 5)
Oesophagus max. width	26 (24–29) (n = 3)
Tail length	69 (64–74) (n = 3)	95	95	158	116 (95–170) (n = 5)
Mid-tail width	27 (25–30) (n = 3)
Tail width at level of cloaca	42 (38–44) (n = 3)
Caudal lappets	3 large (n = 3)			1 small	none (n = 5)
Caudal lappets length	3–6/4–5/5–8 (n = 3)
Left spicule length	267 (252–275) (n = 3)	301	335	285	318 (310–323) (n = 5)
Left spicule handle length	137 (100–163) (n = 3)	151	140	110	164 (150–172) (n = 5)
Right spicule length	91 (70–115) (n = 3)	108	95	90	109 (98–120) (n = 5)
Distance area rugosa to tail tip	No area rugosa (n = 3)	1100–3500	710–3700	950–2500	700–2650 (n = 3)
Area rugosa width	–		17	10
Area rugosa pattern	–	Bosses	Bosses	Bosses	Bosses

Abbreviation: n, number of specimens measured.

Fig. 1.

Litomosa vetuni n. sp. ex Vespertilio murinus (Czech Republic). A-C Female. A Anterior region, lateral view. B Buccal capsule, lateral view. C Posterior region, lateral view. D-F Male. D Caudal region, lateral view. E Left spicule. F Right spicule.

Fig. 2.

Litomosa vetuni n. sp. ex Vespertilio murinus (Czech Republic). Scanning electron micrographs of the anterior extremity. A Female, ventrolateral view of the anterior extremity showing lateral shoulders and a small mouth opening (open arrow) on a slightly elevated areola. B Female, ventrolateral view of the anterior extremity with the mouth opening (open arrow) and one cephalic papilla (solid arrow). C Female, sub-apical view showing the areola with amphids (asterisks) on elevated circular areas and paired cephalic papillae (solid arrows). D Female, close-up of a cephalic papilla with a flattened apical surface. E Male, view of the anterior region showing the excretory pore (triangle) as a shallow depression on the ventral body surface, the apically positioned mouth opening (open arrow), and cephalic papillae situated immediately posterior to it (solid arrow). F Male, lateral view of the anterior extremity showing an amphid (asterisk) and a cephalic papilla (solid arrow).

Fig. 3.

Litomosa vetuni n. sp. ex Vespertilio murinus (Czech Republic). Scanning electron micrographs of the caudal extremity. A Female, ventrolateral view of the posterior extremity showing the anus (triangle) and the median lappet (solid arrow); one lateral lappet is missing, broken off at its base (hollow arrow), while the opposite lateral lappet is obscured by surface debris. B Female, closer view of the posterior extremity showing the relative positions of the median lappet (solid arrow) and one of the lateral lappets (hollow arrow); a small papilla-like structure (cross) is visible anterior to the median lappet, appearing as two to three closely apposed digitiform projections with rounded tips. C Female, detail of the papilla-like structure. D Female, detail of the median lappet. E Female, detail of the base of the broken lateral lappet. F Male, lateral view of the caudal extremity showing the median lappet (solid arrow) in apical position and one of the lateral lappets (solid arrows); a pore-like structure is visible beneath the lateral lappet, interpreted as a phasmid (star). G Male, close-up of the phasmid (star) at higher magnification.

Female. [Based on 6 gravid specimens.] Vulva located at or slightly posterior to the oesophago-intestinal junction (Fig. 1A). Vagina consisting of a proximal transverse tube lined with cuticle, followed by bend and chamber lined with thick epithelium; sphincter located between two bends, leading to ovijector (Fig. 1A). Tail blunt, with 3 conical spine-like lappets positioned just anterior to tip on ventral surface (Fig. 3A and B). Median lappet situated most posteriorly (Fig. 3A, B, D); lateral lappets slightly anterior and symmetrical. Small papilla-like cuticular structure present anterior to median lappet, forming 2 or 3 closely apposed digitiform projections with rounded tips (Fig. 3B and C). Microfilariae folded within sheath; body slightly attenuated from anterior to caudal extremity.

Male. [Based on 3 specimens.] Area rugosa absent in posterior ventral region. Caudal papillae 7: 1 precloacal papilla and 3 pairs of paracloacal papillae, symmetrically arranged. Tail slightly tapering, with 3 blunt spine-like lappets arranged as in females - median lappet more apical, two others lateral and slightly anterior (Fig. 3F and G). Phasmids paired, visible as small pore-like structures beneath lateral lappets (Fig. 3F and G). Spicules unequal in shape and size (Fig. 1D). Left spicule (Fig. 1E) markedly longer than right (Fig. 1F); handle forming more than half of total length, tapering distally into finger-like projection; lamina whip-like, accompanied by delicate membrane extending beyond its tip as flap; distal tapering of handle and lamina base forming forceps-like structure. Right spicule (Fig. 1F) boomerang-shaped, distal part with prominent dorsal heel and terminal knob-like tip.

3.1.3. Remarks

The new species is assigned to Litomosa Yorke and Maplestone, 1926, based on the presence of a complex buccal capsule composed of four well-sclerotised segments, with the posterior segment embedded in the oesophagus, as defined by Bain (1966) and Anderson and Bain (1976). Additional features consistent with the genus are the absence of a cephalic shield, a non-attenuated cephalic extremity, an undivided oesophagus, the vulva located near the end of the oesophagus, a poorly defined separation between the handle and lamina of the left spicule, and a caudal extremity of the female bearing tiny lappets (Anderson and Bain, 1976). Although ventral cuticular ornamentation (area rugosa), considered a typical feature of the genus (Bain, 1966; Anderson and Bain, 1976), was not observed in the present specimens, Martin et al. (2006) noted that this structure is variable in occurrence and absent in some species of Litomosa.

Litomosa vetuni n. sp. belongs to a group of congeners characterised by an enlarged segment III of the buccal capsule and shared basic spicule morphology. The left spicule consists of an elongate, sclerotised handle and a very slender, whip-like lamina, closely accompanied by a delicate membrane that extends beyond the lamina tip to form a flap. The right spicule is markedly shorter, with a prominent dorsal heel near its distal termination. In addition to the type-species, L. filaria (van Beneden, 1872), this group comprises seven other species described from European, African and Malagasy bats belonging to Miniopteridae, Rhinolophidae and Vespertilionidae: L. adami Petit, 1980; L. beshkovi Jancev, 1971; L. chiropterorum Ortlepp, 1932; L. goodmani Martin, Bain, Jouvenet, Raharimanga, Robert and Rousset, 2006; L. ottavianii Lagrange and Bettini, 1948; and L. seurati Martin, Bain, Jouvenet, Raharimanga, Robert and Rousset, 2006 (see Table 1 for a summary of all known Litomosa species, hosts, localities and references).

Of these species, the proportions and shapes of the four segments of the buccal capsule in L. vetuni n. sp. resemble those of L. ottavianii from Miniopterus schreibersii in France, and L. seurati from Rhinolophus ferrumequinum in Algeria, as illustrated by Bain (1966). Litomosa ottavianii was originally reported from M. schreibersii and V. murinus in Italy by Lagrange and Bettini (1948). Bain (1966) revised the genus and described Litomosa beaucournui from nematodes collected from M. schreibersii, R. ferrumequinum and Rhinolophus sp. in France, Algeria and Morocco. Bain (1966) distinguished two groups of specimens from Rhinolophus spp.: those from Pouade (Pyrénées-Orientales, France), which she regarded as typical L. beaucournui, and smaller specimens from Algeria, Morocco and Salin de Badon (Bouches-du-Rhône, France), differing in body size, buccal capsule shape, caudal morphology and width of the area rugosa. She interpreted these differences as host-related polymorphism rather than species-level distinction. Litomosa beaucournui was later recorded in France (Combes and Clerc, 1970) and Bulgaria (Jancev and Stojkova, 1973) from several bat hosts. Subsequently, based on comparisons of the descriptions of L. ottavianii and L. beaucournui, Sonin (1975) concluded that the two species were synonymous. However, Martin et al. (2006) re-examined the Algerian specimens from R. ferrumequinum, which had originally been identified as L. filaria by Seurat (1921) and later interpreted by Bain (1966) as belonging to her newly described species L. beaucournui. They designated these specimens as the type-series of a new species, L. seurati, based on the narrower area rugosa, the tail in female bearing two separate conical spines and the narrower buccal capsule (15 vs 22 μm in L. ottavianii; figure 1B, C in Bain, 1966), which they considered diagnostic features.

However, the diagnosis of Martin et al. (2006) essentially restates the variable features already noted by Bain (1966) and does not include a detailed description, new measurements and drawings or robust evidence for species-level separation. Given the variability highlighted by Bain (1966), the absence of stable diagnostic traits distinguishing L. seurati from L. beaucournui (currently considered a synonym of L. ottavianii), and the lack of comprehensive morphological or molecular analysis, the taxonomic status of L. seurati remains uncertain. Although L. vetuni n. sp. shares a similar position of the nerve-ring and vulva (i.e. just anterior to the oesophageal midpoint and near the oesophago-intestinal junction, respectively), it differs from the two aforementioned species by the following features (see Seurat, 1921; Lagrange and Bettini, 1948; Bain, 1966): (i) a buccal capsule with a triangular segment III exhibiting a rather straight inner surface (vs proportionally larger segment III with a concave inner surface in L. ottavianii, and proportionally smaller and also concave in L. seurati); (ii) a buccal cavity with a posterior third constriction between segments III and IV (vs buccal cavity with a median constriction between segments II and III in L. ottavianii and L. seurati); (iii) tail in the female with three prominent lappets of similar size (vs tail with paired ventrolateral lappets, often bifid, and one or two small median lappets in L. ottavianii, and tail with two large lateral and one small median lappet in L. seurati); (iv) tail in the male bearing three lappets (vs tail with no reported lappets in L. ottavianii, and tail with a small subterminal ventral lappet in L. seurati); (v) area rugosa absent (vs area rugosa composed of cuticular bosses arranged in a broad band in L. ottavianii, and in a narrow band in L. seurati); and (vi) right spicule approximately uniform in width along its entire length, terminating in a knob-like distal tip (vs slightly widened proximally and ending in a spatulate distal end in L. ottavianii and L. seurati).

Among the two nominal species of Litomosa for which molecular data are currently available (L. chiropterorum and L. westi), L. chiropterorum emerges as the closest congener of L. vetuni n. sp. in both cox1 and 12S rDNA phylogenies (see below). Although L. chiropterorum belongs to the same morphological group characterised by an enlarged segment III of the buccal capsule, it is morphologically less similar to L. vetuni n. sp. than the congeners discussed above. Litomosa vetuni n. sp. differs from L. chiropterorum in several characters, including the reduced and poorly differentiated segment II (vs distinctly developed), the triangular and nearly equidimensional segment III (vs broader-than-long), and the presence of three caudal lappets in both sexes (vs two in females and none in males).

Comparative morphometric data for L. vetuni n. sp., the morphologically most similar congeners (L. ottavianii and L. seurati), and the closest sequenced species (L. chiropterorum) are summarized in Table 2, Table 3. Females of L. vetuni n. sp. (20–30 mm) fall within the range reported for L. seurati (19–25 mm), but are distinctly smaller than females of L. ottavianii (42–57 mm) and L. chiropterorum (72 mm). Body length data for males of L. seurati are not available; however, males of L. vetuni n. sp. (12–14 mm) are markedly shorter than those of L. ottavianii (27 mm) and L. chiropterorum (28–41 mm). Males of L. vetuni n. sp. also tend to exhibit a shorter left spicule. The length of the left spicule (252–275 μm) approaches the lower values reported for L. ottavianii (301–335 μm), L. seurati (285 μm) and L. chiropterorum (310–323 μm), whereas the length of the right spicule largely overlaps among all compared species. With respect to the buccal capsule, L. vetuni n. sp. overlaps in size with L. seurati, while L. ottavianii and L. chiropterorum generally possess a larger buccal capsule. Overall, L. vetuni n. sp. shows closest morphometric similarity to L. seurati, while differing in the morphological characters described above.

3.2. Comparative sequence analysis

A total of 30 cox1 sequences (594–689 bp) were generated during our previous studies from the Czech Republic (25 sequences; Pikula et al., 2023; Bednarikova et al., 2025a), Russia (1 sequence; Bednarikova et al., 2025a), Georgia (3 sequences; Bednarikova et al., 2025b), and Armenia (1 sequence; Bednarikova et al., 2025b) (see Supplementary Table S1 for details). Out of these, 25 sequences were unique, and the sequence divergence among them ranged from 0.1 to 1.4%. This intraspecific variability is comparable to that of other related recognised species (e.g. 0.2–1.9% in L. chiropterorum; 0.3–3.7% in Litomosoides brasiliensis). The genetic distance from the closest sequence of another Litomosa species (Litomosa sp., MH411205, ex Hypsugo savii, Bulgaria) ranged from 8.1 to 9.1%.

A total of four 12S rDNA sequences (464–473 bp) were identical in their overlapping regions; a single representative sequence was deposited in GenBank (PV364979). The results of the BLAST analysis showed that the sequence for L. chiropterorum (GenBank: FM209546, ex Miniopterus natalensis, South Africa) was the most similar sequence (92% identity, 82% query cover). The genetic distance of our sequence (PV364979) to the available 12S rDNA sequences of L. chiropterorum ranged from 1.7 to 3.8%.

3.3. Molecular phylogeny

Phylogenetic analyses based on cox1 and 12S rRNA genes placed L. vetuni n. sp. within Litomosa, with all used sequences of the new species forming a monophyletic clade distinct from other included congeners (Fig. 4, Fig. 5 and Supplementary Fig. S1 and S2, respectively). In the cox1 phylogeny, filarial nematodes associated with bats clustered into 3 clades corresponding to Litomosa, Litomosoides, and a putative new genus within the Onchocercidae (Fig. 4; Supplementary Fig. S1). Litomosa was represented by 10, possibly 11, species-level genetic lineages, only three of which corresponded to described species (L. chiropterorum, L. vetuni n. sp., and L. westi). Litomosoides was represented by at least 12 species-level genetic lineages assigned to 11 described species; however, some sequences appear to be misidentified in their original annotations. The putative new genus within Onchocercidae comprised two species-level genetic lineages, both currently undescribed. DNA of one of these lineages was frequently detected in V. murinus and its ectoparasites in our previous study (Bednarikova et al., 2025a). The L. vetuni n. sp. clade included four sequences representing the holotype (OP796371), microfilariae from the holotype's host (OP796369), the paratype (PQ042406), and microfilariae from the paratype's host (PQ042404), and three other sequences (PQ042391, PV023864, and PV023865). All sequences of Litomosa vetuni n. sp. clustered within a single lineage, forming a well-supported monophyletic clade within Litomosa. Higher observed intraspecific variability of several clades (e.g. L. vetuni n. sp. clade or Litomosa spp. clades from Madagascar) is at least partially attributable to the fact that the majority of the sequences come from microfilarial DNA, representing multiple larvae per sample. In such cases, ambiguous bases may occur, and the algorithm of phylogeny assigns a random possible nucleotide in such positions (e.g. W is randomly represented as A or T, regardless of the real combination in individual specimens). This phenomenon can artificially increase the intraspecific diversity observed in phylogeny. The closest related lineage to Litomosa vetuni n. sp. was represented by a single otherwise unpublished sequence obtained from Hypsugo savii in Bulgaria, which is deposited in GenBank as Litomosa sp. However, in the absence of published morphological data, its species identification remains unresolved.

Fig. 4.

Schematic representation of the maximum likelihood phylogenetic tree based on cox1 gene sequences of members of the genera closely related to Litomosa spp. and Litomosoides spp. (A), with a detailed phylogeny of Litomosa spp. for which sequences are available (B). The final length of the alignment was 1560 bp and contained 109 sequences. The tree was constructed using the evolution model TIM3+F+R4. Two sequences of Dipetalonema caudispina were used as the outgroup. The sequences of Litomosa vetuni n. sp. are marked in red. The species-level lineage of a putative new genus of the Onchocercidae frequently detected in Vespertilio murinus in our previous study (Bednarikova et al., 2025a) is marked in blue. Sequences originating from the holotype, the paratype, or tissues of the paratype's host are marked in bold. The scale-bar indicates the number of nucleotide substitutions per site. Only bootstrap values (SH-aLRT/UFB) above the 80/95 thresholds are shown. The sequence labels include the accession number, species, host and country of origin (if available).

Fig. 5.

Schematic representation of the maximum likelihood phylogenetic tree based on 12S rRNA gene sequences of genera closely related to Litomosa spp. and Litomosoides spp. (A), with a detailed phylogeny of species of Litomosa and Litomosoides for which sequences are available (B). The alignment, completed by MAFFT, had a final length of 559 bp and contained 91 sequences (one originating from this study). The tree was constructed using the evolution model TIM3+F+G4. Two sequences of Foleyella furcata were used as the outgroup. The sequence from this study is marked in bold and blue. The scale-bar indicates the number of nucleotide substitutions per site. Bootstrap values (SH-aLRT/UFB) above the 80/95 thresholds are displayed. Sequence labels include the accession number, species, host and country of origin (when available).

The 12S rDNA phylogeny placed the single sequence of Litomosa vetuni n. sp. within Litomosa, together with sequences of only two other Litomosa spp. for which 12S rDNA data are currently available on GenBank, L. westi and L. chiropterorum (Fig. 5; Supplementary Fig. S2). Similarly to the cox1 phylogeny, the 12S rDNA phylogeny resolved Litomosoides into possibly up to 13 species-level genetic lineages corresponding to 12 nominal species, although support for many of the recovered clades was low. As no 12S rDNA sequences of the putative new bat-associated genus within the Onchocercidae were generated in our previous study, this lineage is absent from the 12S rDNA phylogeny.

4. Discussion

Species of Litomosa parasitizing V. murinus reported to date include L. filaria (van Beneden, 1872), L. ottavianii Lagrange & Bettini, 1948 and L. vaucheri Petit, 1980. The specimens described herein as L. vetuni n. sp. increase the number of Litomosa spp. known from this host to four, and represent the first species for which both morphological and molecular characters were combined to support its description. The combination of diagnostic characters distinguishing L. vetuni n. sp. from its morphologically and phylogenetically closest congeners highlights several key features that warrant brief additional discussion.

Although most Litomosa spp. share an undivided oesophagus, a feature reflected in the generic diagnosis (Anderson and Bain, 1976), the presence of a divided oesophagus into distinct muscular and glandular regions in L. aelleni (Tibayrenc et al., 1979) and L. hepatica (Guerrero and Bain, 2011) raises questions about the diagnostic boundaries of the genus. This discrepancy may reflect genuine variation within Litomosa but may equally stem from difficulties in distinguishing the muscular and glandular portions of the oesophagus, which are not always sharply separated. Clarifying such morphological inconsistencies is important for estabilising the generic limits of Litomosa and for interpreting other potentially variable structures, including the area rugosa.

The area rugosa is a ventral ornamentation of the caudal region in males, consisting of cuticular structures such as bosses, beads or ridges arranged in a transverse band. Its morphology, which is species-specific, was first defined by Bain (1966), who introduced the term area rugosa and noted its likely mechanical function during copulation. According to Martin et al. (2006), the area rugosa is absent in four species (L. aelleni, L. americana, L. japonica, and L. miniopteri) and has not been mentioned in several others, most likely due to descriptions being incomplete. No area rugosa was observed in L. vetuni n. sp., despite examination of three males under light microscopy; unfortunately, the SEM images did not clearly capture the region. These findings highlight the need for further investigation of this character, as apparent differences among species may reflect poor preservation or a weakly developed area rugosa that escaped detection.

Spicule morphology and the structure of the buccal capsule represent two of the most important diagnostic characters for distinguishing species of Litomosa, yet both are inconsistently treated in earlier species descriptions. Although spicules are among the key features used in nematode taxonomy (e.g. Musah-Eroje et al., 2021), many earlier descriptions of Litomosa species provide only minimal information or omit spicule morphology altogether (e.g. Ortlepp, 1932; Mackerras, 1962; Bain, 1966), limiting their utility for reliable comparisons among congeners. A similar problem affects the buccal capsule, whose taxonomic importance was emphasised by Bain (1966). According to Bain (1966), it consists of four vertically arranged segments (I-IV) in an antero-posterior direction: segment IV is intra-oesophageal, segment III lies on the oesophageal surface, segment II is usually well defined by two transverse grooves, and segment I is slender with poorly delimited walls. Variation in the proportions and delimitation of these segments, as well as the overall configuration of the buccal cavity, provides reliable characters for species identification, provided that illustrations and measurements are sufficiently detailed and accurate. Because older descriptions are often incomplete or inconsistent, high-quality drawings and measurements of both the buccal capsule and the spicules are essential for accurate comparison among species of Litomosa.

Species of Litomosa appear to be closely related to those of Litomosoides Chandler, 1931, as species in both genera possess a thick buccal capsule embedded posteriorly in the oesophagus and share similar host associations (Guerrero et al., 2002). Both groups comprise numerous species parasitizing bats, with Litomosa occurring mainly in Old World bats and Litomosoides being more diverse in the New World, infecting bats, rodents and marsupials (Junker et al., 2009). The North American species L. thomomydis and L. westi, formerly assigned to Litomosoides but transferred to Litomosa by Guerrero et al. (2002), illustrate the morphological overlap and a close relationship between the two genera within the Onchocercidae (Casiraghi et al., 2004). The two genera are distinguished principally by the morphology of the buccal capsule and by characters of the microfilariae. In species of Litomosa, the buccal capsule is complex and segmented (Bain, 1966), whereas in species of Litomosoides it is simple, tubular and unsegmented (Guerrero et al., 2002). Microfilariae of Litomosoides spp. are typically straight, sheathed and characterised by a prominent cephalic hook, while those of Litomosa spp. are folded within a shorter sheath and bear only a minute hook (Moorhouse et al., 1979; Tibayrenc et al., 1979; Petit, 1980). Although these morphological differences provide a practical basis for separating species of the two genera, the presence of overlapping characters and historically inconsistent species assignments suggest that their diagnostic limits may not be fully resolved.

This uncertainty is compounded by the limited molecular sampling currently available for Litomosa. The cox1 data indicate the presence of 9–10 species-level genetic lineages; however, only three of these correspond to described species (L. westi, L. chiropterorum, and L. vetuni n. sp.) (Casiraghi et al., 2004; Junker et al., 2009; present study), and 12S rDNA sequences are available for these three taxa only, despite the 24 species currently recognised in the genus. A similarly limited molecular coverage affects Litomosoides, for which sequence data are available for 12 out of 32 species listed by Bain et al. (2008). The scarcity of sequence data for both genera underscores the need for an integrative taxonomic approach combining intense molecular sampling with high-quality morphological documentation, which will be essential for resolving their relationships and stabilising the generic limits within this group of filarial nematodes.

Phylogenetic analyses of both cox1 and 12S rDNA placed L. vetuni n. sp. within the Litomosa clade as currently represented in GenBank, while Litomosoides formed a distinct clade in both analyses. However, broader inference of phylogenetic relationships within Litomosa remains extremely limited due to both the scarcity of available sequences and their frequent lack of direct association with morphologically identified material. Currently, molecular data represent only approximately 10 species-level genetic lineages, corresponding to fewer than half of the described species in the genus. Moreover, a substantial proportion of available sequences originates from Africa and Madagascar, regions where only a limited number of described Litomosa species are currently recognised, suggesting that several sequenced lineages may correspond to undescribed taxa, while the majority of described species remain entirely unsampled molecularly (see Table 1). The cox1 phylogeny included up to seven additional unidentified Litomosa lineages and one sequence deposited as Litomosoides sp. that clustered within the Litomosa clade. In this phylogeny, L. chiropterorum and L. vetuni n. sp. were resolved in separate lineages. Without associated morphological vouchers, the taxonomic identity of the unidentified lineages cannot be assessed.

Combined with our previous studies, DNA of L. vetuni n. sp. has been detected in different host-associated organisms across three distinct regions: the Czech Republic, the Caucasus (Armenia), and Western Siberia (Russia) (Pikula et al., 2023; Bednarikova et al., 2025a, 2025b). These data suggest that the distribution range of Litomosa vetuni n. sp. spans at least from central Europe to central Asia and may broadly correspond to the distribution of its type-host, Vespertilio murinus (Rydell and Baagøe, 1994). Our previous data also suggest that L. vetuni n. sp. may not be strictly host-specific, with M. schreibersii, R. ferrumequinum, or potentially other bat species serving as its definitive hosts. Relatively low host specificity has also been reported in other filarial parasites, such as Dirofilaria spp. (Genchi and Kramer, 2020).

The macronyssid mite Steatonyssus spinosus could serve as a vector of L. vetuni n. sp. (Bednarikova et al., 2025a). Although blood-sucking dipteran insects commonly serve as vectors of filarial nematodes (Morchón et al., 2012), several species rely on different types of vectors, such as ticks (Santos et al., 2017) or lice (Ebmer et al., 2022). The hypothesis that S. spinosus may represent an important vector of bat-infecting filarial nematodes is partially supported by experimental studies on the macronyssid mite Ornithonyssus bacoti and Litomosoides sp., which demonstrated that infective larvae of related filarial nematodes are capable of developing within macronyssid mites (Bain et al., 2002).

Interestingly, DNA sequences representing two additional filarial lineages (Onchocercidae gen. sp.) were also detected in our previous studies, with one of these lineages being frequently found in V. murinus and its ectoparasites (Bednarikova et al., 2025a, 2025b). Based on cox1 sequences, both lineages are clearly separated from Litomosa and Litomosoides and may represent a novel genus of bat-infecting nematodes. This finding further highlights the need for a comprehensive revision of bat-associated filarial nematodes using integrated morphological and molecular approaches (Ebmer et al., 2022; Colella et al., 2025).

5. Conclusions

Litomosa vetuni n. sp. is only the third species in the genus for which both morphological and molecular data are available, emphasising the overall scarcity of genetic information for Litomosa spp. Our findings also highlight inconsistencies in previously published descriptions of several diagnostic characters, including the structure of the oesophagus, the configuration of caudal features in both sexes, and the reported presence or absence of an area rugosa. These issues underscore the need for a comprehensive taxonomic revision of bat-associated filarial nematodes, supported by broader molecular sampling and standardised morphological documentation, particularly high-quality illustrations of key structures such as the buccal capsule and spicules. To advance our understanding of these parasites, future studies should adopt integrative approaches that combine morphology, genetics, host and geographic distribution, and vector ecology.

Ethical approval

Ethical approval was not required for the study involving animals, as it was in accordance with local legislation and institutional requirements, since the research involved only cadavers of bats.

CRediT authorship contribution statement

Eva Řehulková: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft. Jiri Pikula: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Funding acquisition, Resources, Supervision, Writing - original draft, Writing - review & editing. Sarka Bednarikova: Validation, Formal analysis, Visualization, Writing - review & editing. Ondrej Danek: Validation, Formal analysis, Visualization, Writing - review & editing. Šárka Mašová: Investigation, Methodology, Visualization, Writing - original draft. Monika Nemcova: Investigation, Writing - review & editing. Vladimir Piacek: Investigation, Writing - review & editing. Katerina Zukalova: Investigation, Writing - review & editing. Jan Zukal: Supervision, Investigation, Writing - review & editing.

Funding

This research was supported through the project FVHE/Pikula/2025ITA22.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary file. The sequences are deposited in the GenBank database under the accession numbers PV364979 (12S rDNA) and OP796363-OP796372, PQ042391-PQ042417, and PV023858-PV023875 (cox1; all details are provided in Supplementary Table S1).