Introduction
Invasive species can alter ecosystems by outcompeting local fauna and causing economic damage (Pejchar & Mooney, Reference Pejchar, Mooney, Perrings, Mooney and Williamson2010; Dunn et al., Reference Dunn, Torchin, Hatcher, Kotanen, Blumenthal, Byers, Coon, Frankel, Holt, Hufbauer, Kanarek, Schierenbeck, Wolfe and Perkins2012; Bellard et al., Reference Bellard, Cassey and Blackburn2016; Essl et al., Reference Essl, Lenzner, Courchamp, Dullinger, Jeschke, Kühn, Leung, Moser, Roura-Pascual and Seebens2019). The success of invasive species in invaded territories is generally explained by reasons such as lack of enemies, ability to adapt the environment to their lifestyle, use of resources other than native species, and other factors (Colautti et al., Reference Colautti, Ricciardi, Grigorovich and MacIsaac2004; Torchin et al., Reference Thomas, Vida, Frisse, Mundo and Baldwin2003; Ross et al., Reference Ross, Ivanova, Severns and Wilson2010; Tomé et al, Reference Tomé, Harris, Perera and Damas-Moreira2021).
Invasive species can also carry pathogens and parasites. Gastropods are known to serve as intermediate hosts for many helminths parasitic in vertebrates (Grewal et al., Reference Grewal, Grewal, Tan and Adams2003). Widespread invasive gastropods such as Achatina (Lissachatina) fulica Férussac, 1821, Parmarion martensi Simroth, 1893, Rumina decollata (Linnaeus, 1758) have been implicated in the transmission of the causative agent of meningoencephalitis, the rat lungworm Angiostrongylus cantonensis (Kim et al., Reference Kim, Hayes, Yeung and Cowie2014; Valente et al., Reference Valente, Del Rosario Robles and Diaz2020; Jaume-Ramis et al., Reference Jaume-Ramis, Alberto Martínez-Ortí, Delgado-Serra, Santiago Mas-Coma, Foronda and Paredes-Esquivel2023). Another invasive slug, Arion vulgaris Moquin-Tandon, 1855, is a carrier of a number of species of strongylid lungworms (Penagos-Tabares et al., 2019). While the importance of the ability to transmit pathogens and parasites harmful to humans and other vertebrates is obvious (Chinchio et al., Reference Chinchio, Crotta, Romeo, Drewe, Guitian and Ferrari2020), it is less clear in the case of pathogens and parasites of the related invertebrate taxa. Can native species of gastropods be affected by the disease transmitted by their alien congeners? Similar to vertebrates, the lack of coevolved resistance to the parasite suggests that spillover of an alien macroparasite to a related native species will be detrimental to the new host (Prenter et al., Reference Prenter, MacNeil, Dick and Dunn2004; McIntire & Juliano, Reference McIntire and Juliano2021; Romeo et al., Reference Romeo, Piscitelli, Santicchia, Martinoli, Ferrari and Wauters2021). Here we consider the nematode taxa that either use gastropods as their sole host or are opportunistically parasitic/pathogenic to their hosts.
In the case of the well-being of land molluscs, the most serious threat will be posed by pathogenic nematodes of the genus Pelliodits (formerly known as Phasmarhabditis), which have the greatest potential impact on their hosts/associates. The genus Pellioditis includes species found in association with gastropods (17 species) as well as earthworms (3 species). Another species, P. huizhouensis (Huang et al., Reference Huang, Ye, Ren and Zhao2015), has been found in decaying plant tissue. The association with gastropods is used to classify the species as necromenic or pathogenic. Different species and strains of Pellioditis differ in their ability to kill their hosts (Rae & Cutler, Reference Rae and Cutler2020; Laznik et al., Reference Laznik, Majić, Trdan, Malan, Pieterse and Ross2020; Schurkman et al., Reference Schurkman, De Ley I and Dillman2022). Rae (Reference Rae2017) reported that snails have defence mechanisms against many parasitic nematodes, including Pellioditis, that result in encapsulation of the nematodes on the inner surface of the shell. There have been no reports of encapsulation mechanisms in slugs. In gastropods, Pellioditis nematodes prefer a mantle cavity habitat. In contrast to gastropod-associated species, earthworm-associated Pellioditis spp. (at least P. pellio and P. pelhamensis) invade the nephridia of their earthworm hosts, where they remain using a necromenic strategy. Earthworms have developed quite strong defences against invertebrate invaders, allowing them to fight off by forming so-called brown bodies. Brown bodies are free-floating, not attached to the body walls, small objects formed by a nematode juvenile or a bristle or a protozoan or some other kind of debris that has entered the host coelom and been enveloped by the host’s secret. Under peristalsis of an earthworm body, brown bodies are driven to the caudal segments of a host, where they remain (Poinar & Hess, Reference Poinar, Hess, Bulla and Cheng1977; Valembois et al., Reference Valembois, Lassègues and Roch1992). Tandingan De Ley et al. (Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023) reported that some isolates of P. pellio and P. pelhamensis caused mortality of earthworms Eisenia spp. and slugs when treated experimentally, while Cutler and Rae (Reference Cutler and Rae2022) found no pathogenic effects of P. californica on Lumbricus terrestris and Eisenia fetida.
The interest in the mollusc-associated Pellioditis is mainly due to its pathogenic effects on pestiferous slugs. This property has been used to replace dangerous chemicals in biological pest methods. There are currently two hermaphroditic species (P. hermaphrodita and P. californica) which have been commercialised and are available in many countries in Europe and North America. The potential of yet other Pellioditis species to kill pestiferous gastropods has been shown experimentally (Mc Donnell et al., Reference Mc Donnell, Colton, Howe and Denver2020; Nermut’ et al., Reference Nermut’, Holley, Ortmayer and Půža2022; Pieterse et al., Reference Pieterse, Haukeland, Půža, Ross and Malan2022).
As emphasised by Tandingan De Ley et al. (Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023), the lethal effect of different Pellioditis spp. on earthworms should be considered before developing a biocontrol product.
Invasive gastropod species can also bring their own parasites to invaded territories, which can affect local species (Ivanova et al., Reference Ivanova, Mazakina and Spiridonov2022; Ivanova, Reference Ivanova2023). There is already evidence of the distribution of commercial strains of Pellioditis aka Phasmarhabditis in many countries, the USA, Canada, South Africa, and New Zealand (Howe et al., Reference Howe, Ha, Colton, De Ley, Rae, Ross, Wilson, Nermut, Zhao, Mc Donnell and Denver2020; Schurkman et al., Reference Schurkman, De Ley I and Dillman2022), which may have arrived with alien gastropods. So, it is therefore likely that gastropods, as well as other travelling and invasive species, keep their nematode associates once they arrive in new territories. Will parasites continue to coexist with their hosts, or will they switch hosts when more desirable hosts become available? Also, they may just expand their host range but remain in the original hosts. Will related native host species be affected by new parasites carried by newly arrived aliens?
A slug species, Krynickillus melanocephalus (Agriolimacidae), was first described by J. Kaleniczenko in the Stavropol region north of the Caucasus (Kaleniczenko, Reference Kaleniczenko1851). Shortly afterwards, it was also recorded in the Caucasus region and on the Crimean Peninsula. Krynickillus melanocephalus also occurs naturally in the north-eastern Turkey and northern Iran. Their typical habitat is the forest and subapical zone, but it spreads to the arid zone along the banks of streams and rivers (Likharev & Wiktor, Reference Likharev and Wiktor1980). In invaded areas, it spreads from gardens into suitable green habitats. This slug species has the ability to form large populations and is known to out-compete native agriolimacids once established (Damyanov & Likharev, Reference Damyanov and Likharev1975; Shikov, Reference Shikov2016). Turóci et al. (Reference Turóci, Fehér, Krízsik and Páll-Gergely2020) point out that the diffuse distribution of K. melanocephalus is more indicative of the human-mediated spread of the species than of a natural expansion of its range.
Since the end of the 20th century, the spread of this species has been recorded in Eastern and Central Europe (Latvia, Ukraine, Estonia, Poland, Belarus, Germany, Sweden, Lithuania, Finland, Slovakia – Borleis, Reference Borleis2018; Bössneck & Feldemann, Reference Bössneck and Feldemann2003; Dreijers et al., Reference Dreijers, Stalažs, Pilāte, Jakubāne and Jundzis2017; Korol & Korniushin, Reference Korol and Kornjusin2002; Gural-Sverlova et al., Reference Gural-Sverlova, Balashov and Gural2009; Maćkiewicz & Borys, Reference Maćkiewicz and Borys2023; Meng & Bössneck, Reference Meng and Bößneck1999; Ostrovsky, Reference Ostrovsky2017; von Proschwitz, Reference von Proschwitz2020; Stalažs et al., Reference Stalažs, Dreijers, Ivinskis, Rimšaitė and Džiugelis2017). Citizen science-based survey in 2020 in Hungary has shown that it is already widespread in the country (Turóci et al., Reference Turóci, Fehér, Krízsik and Páll-Gergely2020). In the Russian Plain (Moscow, Tver, and Novgorod regions), it has been recorded since 2009 (Tappert, Reference Tappert2009).
Although economic damage caused by this slug species has been reported in Latvia (Stalažs et al., 2018), it is unlikely to become a serious pest due to its normal diet of algae, fallen fruit, and fungi (von Proschwitz, Reference von Proschwitz2020).
During the surveys of gastropod-associated nematodes in the Russian part of the North Caucasus carried out by the authors in 2017–2023, this slug species was routinely found in almost all searched sites and was dominant in urban habitats (cities of Pyatigorsk, Kislovodsk, Georgievsk), while less common in the wild (Adygea).
The Caucasus Biodiversity Hotspot, which includes the territories of Armenia, Azerbaijan, Georgia, Turkey, Iran, and parts of Russia, is particularly rich in the terrestrial gastropod fauna (Walther et al., Reference Walther, Kijashko, Harutyunova, Mumladze, Neiber and Hausdorf2014). In its turn, the gastropods of the region host quite a number of nematodes from the genus Pellioditis (Dougherty, Reference Dougherty, Dayal and Suresh1953) (senior synonym of Phasmarhabditis Andrássy, 1976) – 5 out of 21 species constituting the genus.
Three new species of Pellioditis were described by the authors from the Russian part of the North Caucasus (Ivanova et al., Reference Ivanova, Geraskina and Spiridonov2020; Ivanova & Spiridonov, Reference Ivanova2023). Yet two new species were found in the Republic of Georgia, which is situated on the other side of the Caucasian range (Ivanova et al., Reference Ivanova, Gorgadze, Lortkhipanidze and Spiridonov2021; Gorgadze et al., Reference Gorgadze, Troccoli, Fanelli, Tarasco and Luca2022). These five Pellioditis species were found to belong to the specific subclade (Pellio group according to Tandingan De Ley et al., Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023) within the genus Pellioditis (Ivanova & Spiridonov, Reference Ivanova2023). The new species of Pellioditis described here was found in Kislovodsk, also in the North Caucasus. Molecular phylogenetic analysis of the new species has shown that it is related to the different clade within Pellioditis which contains gastropod-associated species of European origin as P. papillosa (Schneider, Reference Schneider1866), P. apuliae (Nermut’ et al., Reference Nermut’, Půža and Mráček2016a), P. bonaquensis Footnote 1 (Nermut’ et al., Reference Nermut’, Půža, Mekete and Mráček2016b), two strains of P. californica from USA (Pellioditis ITD235 and ITD046), as well the American P. pelhamensis (Tandingan De Ley et al., Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023) isolated from earthworms (Papillosa group in Tandingan De Ley et al., Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023).
Previous studies have shown that K. melanocephalus has served as a host for several Pellioditis species of Caucasian origin (P. circassica, P. clausiliiae, and P. akhaldaba) (Ivanova & Spiridonov, Reference Ivanova and Spiridonov2024; own unpublished data). Korol and Kornjushin (Reference Korol and Kornjusin2002) reported the presence of nematode species other than Pellioditis, trematode and cestode parasites in K. melanocephalus in Crimea and Kyiv. This demonstrates the ability of K. melanocephalus to play a role in the transmission of various gastropod-associated parasites. Investigation of the consequences of its distribution outside its natural range should include the parasitological studies of the species and related gastropod taxa.
Materials and methods
Nematode material
The new species was isolated from slugs Krynickillus melanocephalus (Kaleniczenko, Reference Kaleniczenko1851) and snails Oxychilus sp. in the city of Kislovodsk. Live gastropods collected on streets and parks of the city of Kislovodsk were brought to the lab and identified by dissection and morphological examination. Extracted parasites were preserved for morphological and molecular studies by preservation in a hot 4–5% formalin and freezing for molecular work. Adult stages of Pellioditis were obtained by growing juveniles on nutritive medium (preliminary frozen gastropod meat or pig kidney).
Nematodes preserved in formalin were processed to anhydrous glycerin for light microscopy as described by Seinhorst (Reference Seinhorst1959). Light microscopic studies and drawings were done using Nikon Eclipse 200 microscope equipped with a drawing attachment. Scale bars are given in micrometres (μm). Abbreviations: V% – distance from anterior extremity to vulva to body length in %; a, b, c – de Man indices. Illustrations were prepared using WACOM Intuos A4 USB drawing tablet (Wacom, Kazo, Japan) and Adobe Illustrator CS5. For the SEM studies, formalin-preserved material was dehydrated in a graded ethanol series, critical point dried using an HCP-2 HITACHI dryer, mounted on aluminium stubs, and coated with gold in a BIO-RAD SC502 sputter coater. Images were taken on a Mira 3Tescan (Brno, Czech Republic).
Molecular characterisation and phylogenetic analysis
The DNA extraction from collected nematodes was done with QIAamp® DNA Mini Kit, Qiagen, Hilden, Germany, according to the producer’s protocol. An ITS rDNA region was amplified with so-called Curran primers TW81: 5′-GTT TCC GTA GGT GAA CCT GC-3′ and AB28: 5′-ATA TGC TTA AGT TCA GCG GGT-3′ (Joyce et al., Reference Joyce, Reid, Driver, Curran, Burnell, R-U and Masson1994). PCR cycling parameters were: primary denaturation at 95°C for 5 min followed by 35 cycles of denaturation under 94°C for 45 s, annealing under 56°C for 1 min, and elongation under 72°C for 60 s, with final elongation at 72°C for 5 min. As direct sequencing of this ITS product was impossible because of multiple peaks, the product was cloned into a pGEM-T vector and transformed into JM109 High Efficiency Competent Cells (Promega®, Leiden, The Netherlands) according to the instructions of the manufacturer. PCR products from clones were sequenced with the same primers as used for primary amplification (TW81 and AB28). Six sequences for clones were obtained, which were representing two main sequence variants. Obtained sequences for these two clones were deposited in NCBI Genbank as PV565434 and PV565435. The sequence of cytochrome c oxidase subunit I of mitochondrial DNA was obtained with two pairs of primers: COI_F1 (5′-CCT ACT ATG ATT GGT GGT TTT GGT AAT TG-3′) and COI_R2 (5′-GTA GCA GCA GTA AAA TAA GCA CG-3′) proposed by Kanzaki and Futai (Reference Kanzaki and Futai2002) and JB3_F (5′-TTT TTT GGG CAT CCT GAG GTT TAT-3′) и JB7GED_R (5′-ATC AGG ATA ATC CAA ATA YTT WCG WGG-3′) proposed by Bowles et al. (Reference Bowles, Blair and McManus1992) and Derycke et al. (Reference Derycke, Vanaverbeke, Rigaux, Backeljau and Moens2010). The amplification protocol for these two pairs of primers was: primary denaturation – followed by 35 cycles (denaturation at 94°C for 45 sec., annealing for 48°C or 52°C for 45 sec, correspondingly, and elongation under 72°C for 70 sec) with final elongation for 5 min. Final Cox1 mtDNA sequence was assembled from two overlapping sequences obtained with these pairs of primers. Obtained Cox1 mtDNA sequence was deposited in NCBI Genbank as PX118929.
Partial 28S rRNA gene sequences were amplified with LSU391 (5′-AGC GGA GGA AAA GAA ACT AA-3′) (Nadler et al., 2000) and LSU501 (5′-TCG GAA GGA ACC AGC TAC TA-3′) (Thomas et al., Reference Thomas, Vida, Frisse, Mundo and Baldwin1997) primers. The following protocol was used: initial denaturation at 94°C (4 min); 35 cycles of 30 s at 94°C; 30 s at 56°C; 30 s at 72°C; and 5 min at 72°C for the final extension. Obtained LSU rDNA sequence was deposited in NCBI Genbank as PX126001.
For comparative purposes and the phylogeny construction, some sequences from GenBank have been used. Sequence alignments were generated using Clustal_X (Thompson et al., Reference Thompson, Gibson, Plewniak, Jeanmougin and Higgins1997) under default values for gap opening and gap extension penalties. All alignments were analysed using Mega X (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018) for maximum parsimony (MP), neighbour joining (NJ), and maximum likelihood (ML) methods. Because of numerous insertion-deletion events in the ITS rDNA region of Pellioditis nematodes classical MP analysis with PAUP (Swofford, Reference Swofford1998) with gaps treated as a ‘fifth nucleotide’ was also implemented. The evolutionary model was estimated with the help of jModeltest 2.1.7 (Darriba et al., Reference Darriba, Taboada, Doallo and Posada2012). The GTR+G+I as the best-fitted model was estimated with the Bayesian Information Criterion. Bayesian Inference analyses were performed in MrBayes 3.2.7a (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012) at CIPRES (Miller et al. Reference Miller, Pfeiffer and Schwartz2010) portal for 15,000,000 generations, and the first 25% generations were discarded for burn-in. Bayesian Analysis (BA) was performed on IQ-TREE web server (Trifinopoulos et al. Reference Trifinopoulos, Nguyen, von Haeseler and Minh2016).
Results
Description of Pellioditis ciscaucasica Ivanova and Spiridonov, n. sp.
Measurements
Morphometric data on adults and dauer juveniles of P. ciscaucasica n. sp. are provided in Table 1.
Morphometric data of Pellioditis ciscaucasica n. sp. All measurements are in μm and in the form: mean ± S.D. (range)
Description
Adults. Male ca 1.7 mm and female ca 2.3 mm long, body robust, straight when relaxed, slightly tapering to anterior. Cuticle ca 1 μm thick, bearing rows of transverse and longitudinal striations. Longitudinal striations appearing at some distance from anterior, usually posterior to 20th annulus. Lateral field ca 5–7 μm wide consisting of three central, nearly flat, situated side-by-side bands. Each band bearing four central, fine longitudinal striae. Margins of lateral field bordered by 1–2 coarse longitudinal striae (Figure 2D, J, K). Deirids located slightly anterior to excretory pore level. Structure tentatively identified as postdeirid, located in the lateral field in one female specimen, approximately 2 μm posterior to deirid (Figure 2E). Head truncate, lip region short, not offset. Six lips grouped in pairs, each lip bearing a small, short labial papilla in apical position. Each sublateral lip bearing small cephalic papilla located slightly posterior to labial one. Pore-like amphids situated close to labial papillae on lateral lips (Figures 2B–C, 3A–C). Mouth aperture triangular (Figures 2A–C, 3A–C). Stoma tubular, wide, and short (about as long as lip region diam.). Cheilostom not cuticularised. Gymnostom walls parallel, thickened. Stegostom with glottoid apparatus, isomorphic, isotropic, metarhabdions thickened, with three minute warts each. Pharyngeal collar narrow, covering two-thirds of stoma length (Figure 1A–D). Pharynx muscular, differentiated into corpus expanded posteriorly (median bulb), stout, well defined isthmus, and small, pear-shaped terminal bulb as wide as metacorpal expansion or slightly narrower. Terminal bulb with valve and haustrulum. Nerve ring surrounding middle to posterior region of isthmus. Excretory pore position variable, situated from just posterior to nerve ring to level with pharynx base, often obscure (Figure 1G, I). Excretory duct short, weak. Cheilostom, gymnostom, and stegostom mean lengths 4–5 μm, 5 μm, and 10 μm, respectively. Cardia prominent, projecting into intestine. Intestine well developed, forming proventriculus at anterior. Rectal glands present.
Pellioditis ciscaucasica n. sp. A, C, I, J, O–T: female; (A) head, lateral view; (C) head, ventral view; (I) pharynx; (J) spermatheca; (O–P) vulva region, lateral view; (Q) tail, lateral view; (R–T) tail region, ventral view. B, D, G, K–N: male; (B) head, lateral view; (D) head, ventral view; (J) pharynx; (K) tail, subventral view; (L) tail, lateral view; (M, N) spicules. E, F, H, U–W: infective juvenile; (E) head of ensheathed juvenile; (F) head of exsheathed juvenile; (U) posterior of pharynx region; (V) tail of exsheathed juvenile; (W) tail of ensheathed juvenile. (Scale bars in μm.)
Female. Body robust. Lateral fields extending to end of rounded part of tail terminating anterior to phasmids. Stoma 20 μm long on average. Corpus corresponding to 58.3 (52.1–65.4)% of pharynx length. Oviparous. Gonads amphidelphic, ovaries reflexed on dorsal side. Anterior and posterior ovary branches equally long. Oocytes rounded, large, and arranged in two or three rows. Oviduct short. Spermathecae filled with sperm cells (Figure 1J). Uterus spacious, containing 20–30 eggs with thin, smooth shells. Vagina straight, muscular, less than half corresponding body diam. long. Vulva median, a wide transverse slit with prominent margins. Vulval lips flat in mature specimens and inflated in younger ones (Figures 1O, P, 2F, G). Massive copulatory plug over vulva present in fertilised specimens. Rectum inflated, about corresponding body diam. long. Anus an arcuate slit. Tail cupola-shaped with a slender, pointed tip corresponding to ca 55–60% of total tail length (Figure 2L). Phasmids long, projecting, located at tip base (Figures 1Q–T, 2H, I).
Pellioditis ciscaucasica n. sp. SEM images. Female. (A–C) head; (D, J, & K) lateral field; (E) deirid and postdeirid (arrows); (F & G) vulva region, lateral and ventral view, respectively; (H & I) tail region showing phasmids (arrows); (L) tail region sublateral view (arrow indicating phasmid). Abbreviations: a, amphid; cp, cephalic papilla; lp, lip papilla. (Scale bars in μm.)
Male. Shorter and slimmer than females. Lateral field extending to bursa level. Cheilostom, gymnostom, and stegostom mean lengths as in females; mean stoma length 19 μm. Corpus corresponding to 58.9 (53–65)% of pharynx length. Excretory pore poorly conspicuous, situated just posterior to isthmus. Spermatocytes rounded, arranged in two rows. Vas deferens wide, filled with large immature sperm cells ca 7 × 5 μm in size, ejaculatory duct separating from the latter by constriction. Single testis reflexed at ca two body diam. from anterior end. Sperm cells ca 4–6 μm in diam. Bursa open, peloderan. Nine pairs of pedunculate genital papillae (GP) incorporated into bursa (formula 1 + 1 + 1 + 2/1 + 3 + ph). GP 5 and 8 longest; GP 5 and 9 opening dorsally. GP 6–7 and GP 9 not reaching edge of bursa (Figures 1K, L, 3D–F). Tail tip reaching edge of bursa (Figure 3F). Phasmids papilliform, prominent, situated posterior to GP 9 and opened on ventral surface of bursa (Figure 3J, K). Three pericloacal papillae present: precloacal papilla located on anterior cloacal lip in median or submedian position and two process-like papillae situated at lateral margins of cloacal opening (Figure 3F). Spicules nearly straight, uniformly thick for most of spicule length, tapering to tips, and about twice longer than the corresponding body diam., with manubria ca 4.5 μm long and 3 μm wide (Figure 3F–H). Velum indiscernible. Distal tip pointed, with a distinct hole (Figures 1M, N, 3I, L). Gubernaculum boat-shaped, about half as long as spicules; dorsal and ventral processes not observed (Figure 3G).
Pellioditis ciscaucasica n. sp. SEM images. Male. (A–C) head, subapical view, arrows indicating lip and cephalic papillae and amphids; (D) tail, lateral view; (E) tail, ventral view; (F–G) postcloacal region of tail, ventral view, arrow indicating gubernaculum; (H) precloacal region of tail, arrow indicating ventral median papilla; (I) distal tips of spicules, dorsal view, arrow indicating hole at spicule tip; (J–K) posteriormost bursa margin, ventral view, arrows indicating phasmids; (L) distal tips of spicules, ventral view, arrow indicating hole at spicule tip. (Scale bars in μm.)
Dauer juvenile (ensheathed). Body straight when heat-killed, slender, tapering gently towards head end. Cuticle loose, transversally and longitudinally striated; longitudinal striations appearing posterior to 20–22nd annulus (Figure 4A, B). Lateral field with nine, low, even ridges (Figure 4H). Head rounded, distinct apical cuticular cap present (often inverted), lip region low, very slightly offset from body contour, not divided into lips; six cephalic papillae, amphidial apertures situated slightly posterior to circle of cephalic papillae. Mouth aperture enclosed (Figure 4D, E, G). Stoma long, narrow; promesorhabdions slightly curved inwards at both ends (Figure 1E). Pharynx comprising straight, narrow corpus with the slightest metacorpal expansion, isthmus narrow, half as long as corpus and pear-shaped, valvate basal bulb only slightly wider than metacorpal expansion. Nerve ring surrounding isthmus. Cardia prominent, elongated, protruding into intestine. Excretory pore poorly discernible (Figure 1H). Deirids indistinct (Figure 4K). Intestine walls filled with fat globules, lumen with numerous bacterial cells. Genital primordium short, located at mid-body. Tail elongate conoid with filamentous terminus (Figure 1W). Phasmids pore-like, situated anterior to mid-tail (Figure 4J).
Pellioditis ciscaucasica n. sp. SEM images. Infective juveniles. (A–C) anterior region (A, ensheathed juvenile; B, molting juvenile; C, exsheathed juvenile); (D–G & I) head (D, E, & G, ensheathed juvenile; F, E, exsheathed juvenile); (H, L) lateral field of ensheathed juvenile at level of deirid. Arrows indicating (F, G, & I) amphid; (D & E) cephalic papilla; (J) phasmid; (K) deirid. (Scale bars in μm.)
Dauer juvenile (exsheathed). Similar to ensheathed dauer juvenile in internal morphology. Cuticle without longitudinal striations (Figure 4C). Lateral fields with slightly sunken central band ca 3 μm wide flanked by protruding ridges (Figure 4L). Lip region continuous with body contour, divided into six lip sectors, prominent amphid openings at base of lateral lip sectors, six papillae on top of lips (Figures 1F, 4F, I). Excretory pore anterior to basal bulb (Figure 1U). Tail conical, half as long as in ensheathed juvenile, tip rounded. Genital primordium well developed, much longer than in ensheathed juvenile. Phasmids prominent, situated at mid-tail (Figure 1V).
Taxonomic summary
Type material. Holotype female on the slide accession number 1397, two paratype females and two males on the slide accession number 1398, three paratype infective juveniles on the slide accession number 1399 are deposited in the Museum of the Helminthological Collections of the Centre of Parasitology at the Severtsov Institute of Ecology and Evolution, Moscow, Russian Federation.
Type host. Krynickillus melanocephalus.
Other host. Oxychillus (translucidus?).
Prevalence. Sixteen out of 27 gastropod specimens infected (59.3%) by 2–85 nematodes each.
Type locality. Kislovodsk city 43°54′09″ N 42°43′08″.
Etymology. The specific epithet reflects the type locality of the area.
Diagnosis and relationships
The new species is characterised by its morphology and unique DNA sequences. The females possess cupola-shaped tails av. 117 μm long with long pointed terminus and prominent phasmids at the base of the terminus; males have spicules av. 65 μm long with holes at its tips and papilliform phasmids located near tail tip; both adults have lateral fields of three rather wide, nearly flat, longitudinally striated ridges. Infective (ensheathed) juveniles are around 826 μm long and with lateral fields consisting of 9 ridges (10 incisions).
According to the latest revision by Tandingan De Ley et al. (Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023), the genus Pellioditis accounts 21 species.
By its molecular characteristics, the new species is related to the group of species comprising P. papillosa, P. apuliae, P. bonaquensis, P. californica, and P. pelhamensis. These five species and P. safricana (the latter not included in our analysis due to lack of ITS sequence) are treated as the Papillosa group in Tandingan De Ley et al. (Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023). Within this clade/group, P. papillosa, P. bonaquensis, and P. safricana share a similar morphological trait of a cupola-shaped female tail, while the rest of species are characterised by conical female tails.
From P. papillosa, the present species differs in having a different pattern of a lateral field: three bands (= ridges) vs. 5 ridges in adults and 9 ridges (10 incisions) vs. 8 ridges (9 incisures) in infective juveniles and larger body size of females (1191–3384 μm vs. 1476–1923 μm) while having only slightly shorter infective juveniles (826 μm vs. 864 μm grown in vitro and 900 μm in vivo).
By morphological and morphometrical characteristics, the present species is closest to P. bonaquensis but can be distinguished by having the smaller mean spicule length (65 μm vs. 77 μm) and slightly shorter IJs (826 (630–985) μm vs. 902 (808–1050) μm).
Another species of the same group and possessing cupola-shaped female tails, P. safricana, is smaller than P. ciscaucasica n. sp., with remarkably shorter infective juveniles (543 μm). Moreover, infective juveniles of P. safricana possess the unique feature of a cephalic tooth. Female tails of P. safricana are much shorter (46–87 μm vs. av. 117 μm) and bear barely protruding vs. protruding phasmids.
From the rest of Papillosa group members, the new species differs in the shape of the female tail.
Additionally, it can be distinguished from P. apuliae by shorter spicules (65 μm vs. 81 μm and 73 μm, BAR and GRA strains, respectively), the longer male stoma (19 μm vs. 16–17 μm), and male tail (35 μm vs. 56–59 μm). Body length of infective juveniles of BAR and GRA strains of P. apuliae is respectively 812 μm and 986 μm, and the latter value significantly exceeds that in P. ciscaucasica n. sp.
Pellioditis ciscaucasica n. sp. differs from P. californica by gonochoristic vs. hermaphroditic way of reproduction; its females can be differentiated in having larger body size (av. 2296 μm vs. 1501 μm), rounded vs. conoid female tail bearing stronger protruding phasmids, and a less number of ridges (3 vs. 5) in the lateral field.
Apart from the difference in female tail shapes, the new species differs from P. pelhamensis in the larger body size (av. 2296 μm and 1718 μm vs. 1541 μm and 1155 μm, females and males), longer stomas (vs. 17.7 μm and 16.5 μm); slightly longer (65 μm vs. 60.4 μm) and holed vs. bifurcated spicule tips.
The rest of Pellioditis species, which were characterised molecularly belong to different clades within the genus.
Of them, three species can be compared to P. ciscaucasica n. sp. on the basis of a female tail shape, i.e., P. huizhouensis (Huang et al., Reference Huang, Ye, Ren and Zhao2015), P. meridionalis (Ivanova & Spiridonov, Reference Ivanova and Spiridonov2017) of the same clade, and P. zhejiangensis (Zhang & Liu, Reference C-N and Q-Z2020) of the Pellio clade/group.
The present species and P. huizhouensis are also similar in having a similar pattern of the lateral field, spicule length (65 μm and 70 μm, respectively), and prominent phasmids, but are distinguished in having holed vs. indented spicule tips and more sturdy and longer tail spike in females. The present species and P. meridionalis both have infective juveniles of similar length (826 μm and 839 μm), but adult stages of P. ciscaucasica n. sp. are remarkably larger (2296 μm and 1718 μm vs. 1612 μm and 1317 μm, females and males accordingly). Also, males of P. ciscaucasica n. sp. have shorter spicules (65 μm vs. 76 μm) with holes at spicule tips, which were not recorded in P. meridionalis. Females of the new species are characterised by less rounded tail, more robust terminus (not filamentous), and more distinct phasmids than in P. meridionalis.
From P. zhejangensis the present species differs by the longer (65 μm vs. 58 μm) holed male spicules; the more anterior position of an excretory pore (which is never situated posterior to pharynx base as in P. zhejangensis); longer and less rounded female tails with sturdier spikes; significantly longer infective juveniles (826 μm vs. 610 μm) with different lateral field pattern of 9 ridges (10 incisions) vs. 2 ridges (3 incisions).
All other Pellioditis species are characterised by females with a conical or conoid tail gradually tapered to a pointed terminus, thus distinguishing them from P. ciscaucasica n. sp. Additionally, unique DNA sequences help to distinction of the species. At the same time, some of them share certain traits with the new species. Thus, the Caucasus-originated species P. circassica (Ivanova et al., Reference Ivanova, Geraskina and Spiridonov2020), P. clausiliiae (Ivanova et al., Reference Ivanova, Geraskina and Spiridonov2020), P. akhaldaba (Ivanova et al., Reference Ivanova, Gorgadze, Lortkhipanidze and Spiridonov2021), P. thermalis (Ivanova & Spiridonov, Reference Ivanova2023), and P. villasmundi (Ivanova et al., Reference Ivanova2023) from Sicily share the trait of holed spicules with P. ciscaucasica n. sp.
Apart from the different female tail shape, the new species can be differentiated from P. villasmundi by significantly shorter spicules (65 μm vs. 82 μm) and longer IJs (826 μm vs. 773 μm).
Female tail shapes of two strains of the Caucasian P. akhaldaba and P. thermalis are intermediary between a conoid and cupola-shaped ones as an anterior part of the tail is usually broadly conical, and posterior one is subulate. One of the strains of P. akhaldaba, ‘slow’, has females with more rounded tails similar to those in P. ciscaucasica n. sp., but females of the new species can be distinguished from the former in the position of phasmids at the base of a terminus vs. on a terminus. Female tail shape of the strain sindicae of P. thermalis also resembles that of P. ciscaucasica n. sp. but differs in its mean length (102 μm vs. 117 μm).
Compared to Caucasian Pellioditis spp., the present species has the longest IJs but for P. akhaldaba ‘quick’ (871 μm) and Moscow strain (897 μm): 826 μm vs. 742 μm in P. clausiliiae, 702 μm in P. akhaldaba ‘slow’, 670 μm in P. thesamica (Gorgadze et al., Reference Gorgadze, Troccoli, Fanelli, Tarasco and Luca2022, and 717–771 μm in both strains of P. thermalis. Yet one Caucasian Pellioditis, P. thesamica, is not characterised by holes at spicule tips.
Brief descriptions of P. incilaria (Yokoo & Shinohara in Shinohara & Yokoo, Reference Shinohara and Yokoo1958) and P. mairei (Maupas, Reference Maupas1919) allow to distinguish them from the rest of the genus on the basis of a single trait only, i.e., the larger number of genital papillae and large-sized females, respectively. In addition to the difference in the female tail shape, all above said species can be distinguished by the absence of holes at spicule tips.
Molecular characterization and phylogeny of P. ciscaucasica n. sp.
Sequences for the ITS region of DNA fragments are deposited at the NCBI Genbank under numbers PV565434 and PV565435. The 722 bp long ITS rDNA alignment contained 308 constant characters, 122 variable parsimony-uninformative, and 292 parsimony-informative characters. In all three methods of MEGAX analysis, P. ciscaucasica was clustered with other species of the Papillosa-group (data not shown). Notably, support for this clade was visibly stronger when the ‘gaps as fifth nucleotide’ option was used in the MP analysis in PAUP (Figure 5). Bayesian analysis showed strong support for this clade (Figure 6).
Phylogenetic relationships of Pellioditis ciscaucasica n. sp. as inferred from analysis of partial sequence of ITS rDNA. Maximum parsimony analysis with gaps treated as ‘fifth nucleotide’. Bootstrap support values are indicated near the nodes.
Phylogenetic relationships of Pellioditis ciscaucasica n. sp. inferred from Bayesian analysis of partial sequence of ITS rDNA. GTR+G+I evolutionary model. Posterior probability values are indicated near the nodes.
BLAST search for the obtained 1059 bp long Cox1 mtDNA sequence has revealed several related Pellioditis (syn. Phasmarhabditis) sequences. As the various pair of primers were used to obtain different Cox1 mtDNA sequences, their length was unequal, and only six ones were used for analysis with the about 450 bp long alignment for the entire set. No strongly supported affinities of P. ciscaucasica n. sp. with the species in comparison were detected (data not shown). The greatest nucleotide differences (42 bp per 450 bp long alignment) were observed for P. bonaquensis. It seems that as in case with Cox1 mtDNA sequences, different pairs of primers were used by the different authors to obtain LSU sequences of Pellioditis species, which resulted in quite short alignment length (about 400 bp) for the representative set of species. Observed level of nucleotide differences between Pellioditis species was quite low (less than 20 per 400 bp alignment), and the relationships between species were not resolved in phylogenetic trees (data not shown).
Discussion
According to the recent research (Tandingan De Ley et al., Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023), there are two large clades in Pellioditis. The clade united all Papillosa-related species includes P. papillosa, P. apuliae, P. safricana, P. bonaquensis, P. californica, P. pelhamensis, and some undescribed species. The Pellio group/clade contains P. pellio; P. akhaldaba and P. circassica united in a subclade; P. neopapillosa (Mengert in Osche, Reference Osche1952) and P. hermaphrodita (Schneider, Reference Schneider1859) Andrássy, Reference Andrássy1983 + P. villasmundi in another one; P. clausiliiae along with P. thesamica in yet one; and P. zhejiangensis. The authors also recognised the Kenyensis group with P. kenyensis Footnote 2 (Pieterse et al., Reference Pieterse, Rowson, Tiedt, Malan, Haukeland and Ross2020), two South African undescribed species and P. quinamensis (Ivanova & Spiridonov, Reference Ivanova, Mazakina and Spiridonov2022) from Vietnam, and a clade of P. huizhouensis and P. meridionalis. Azzam (Reference Azzam2023), when describing P. eagyptiaca (Azzam, Reference Azzam2023), has shown that P. tawfiki (Azzam, Reference Azzam2003) and P. eagyptiaca were related to P. hermaphrodita and P. neopapillosa, respectively, thus belonging to Pellio group of Tandingan De Ley et al. (Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023).
In the analysis of Tandingan De Ley et al. (Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023), all Pellioditis species discovered in the Caucasian region before the discovery of P. ciscaucasica n. sp. were placed in the Pellio group. In the analysis of Ivanova and Spiridonov (Reference Ivanova2023), which generally agreed with the latter, two smaller subclades were recognised, one uniting circassica-related species and another clausiliiae-related.
The analysis in the present study agrees with that of Tandingan De Ley et al. (Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023) with respect to the clades uniting the species only of non-European origin.
The ITS-based analyses in the present study have placed P. ciscaucasica n. sp. into the Papillosa group and closest to P. pelhamensis. It has also been shown that P. papillosa was most closely related to P. apuliae, while their connection with P. californica was supported weakly. Pellioditis bonaquensis formed a separated branch. In both analyses (Tandingan De Ley et al., Reference De Ley I, Kiontke, Bert, Sudhaus and Fitch2023, and ours), P. pelhamensis was in the basal position. In morphological terms, these two species are easily differentiated based on adult’s morphology, for infective juveniles of P. pelhamensis were not described. Noteworthy that P. pelhamensis is the only associate of earthworms within the Papillosa group of species. The ITS rDNA region of some Pellioditis species can be successfully studied by direct sequencing of the primary PCR products. This is not the case for P. ciscaucasica and some other Caucasian species, where two or more ITS haplotypes can be obtained from a single nematode specimen. Previously, we suggested that such diversity in the ITS rDNA region could be explained by the reunification of the previously isolated populations of mollusc hosts and associated Pellioditis nematodes (Ivanova & Spiridonov, Reference Ivanova and Spiridonov2024).
Although Pellioditis papillosa (syn. Phasmarhabditis papillosa) was one of the first rhabditid species which were described in association with terrestrial molluscs it was relatively rarely found in Europe (Mengert, Reference Mengert1953; Laznik et al., Reference Laznik, Majić, Trdan, Malan, Pieterse and Ross2020), infecting several agriolimacid and arionid slug species. This nematode species was also discovered in the USA (Tandingan De Ley et al., Reference De Ley I, Holovachov, McDonnell, Bert, Paine and De Ley2016) and South Africa (Pieterse et al., Reference Pieterse, Malan, Kruitbos, Sirgel and Ross2017) during surveys for mollusc-associated nematodes. Pellioditis ciscaucasica n. sp. is the only species of the Papillosa group in the Caucasus but shares a number of morphological characteristics with other Caucasian Pellioditis of Pellio group (holes at spicule tips and infective juveniles 700–900 μm long).
The Kislovodsk Nature Park occupies a large territory of almost 10 square km covering the slopes of the Dzhinal Ridge in the city of Kislovodsk, Stavropol Krai, in the North Caucasus. The park began in 1827 with the planting of a promenade of lime trees. Then many alien tree species were imported and planted using non-local fertile soil (chernozem). Similarly, alien gastropod species may have been introduced along with plants and soils. This may partly explain the unrelatedness of the new Pellioditis species to other congeners of Caucasian origin that evolved in non-artificial environments. The gastropod fauna of the Kislovodsk Nature Park is influenced by anthropogenic impacts and is similar to the gastropod fauna of the Pontic-Caspian ecoregion.
In the Caucasus, Krynickillus melanocephalus has been found to be infected by other local Pellioditis species (P. circassica in Pyatigorsk and P. clausiliiae in Georgievsk). Recently, it has been shown that K. melanocephalus introduced into the Main Botanical Garden in Moscow was infected with P. akhaldaba, which originated from Georgia (Ivanova & Spiridonov, Reference Ivanova and Spiridonov2024). This nematode species was found in three alien gastropods, all of the Caucasian origin (K. melanocephalus, Deroceras caucasicum, and Boetgerilla pallens), while the local agriolimacid slug species D. reticulatum (which was recorded as the type host of P. akhaldaba in the Republic of Georgia) was free of infection. At the same time, the spread of this Pellioditis species to another suitable host can be expected, similarly to the case with spillover of ectoparasitic nematode Alloionema appendiculatum from invasive Arion vulgaris to local Arion species in the Main Botanical Garden in Moscow (Ivanova, Reference Ivanova2023). The low host specificity of Pellioditis species and their general necromenic strategy allow them to rely on a wide range of hosts. At the same time, co-introduced parasites may be more pathogenic to the new local hosts.
Thus, the research shows that K. melanocephalus is susceptible to Pellioditis invasion and at the same time can be its vector (Ivanova & Spiridonov, Reference Ivanova and Spiridonov2024). In invaded areas, it also participates in the transmission of brachylaimid trematodes (Korol & Kornjushin, Reference Korol and Kornjusin2002; own observations) and a cestode Molluscotaenia crassiscolex (von Linstow, 1890) together with local gastropod species (Korol & Kornjushin, Reference Korol and Kornjusin2002). Such a high capacity of this slug species to host a wide range of parasites and transmit them to invaded territories may pose a threat to the local gastropod fauna.
Krynickillus melanocephalus appearing in new areas could have come from different habitats where they can be infected by different species of Pellioditis. Primarily, they will be of Caucasian origin. Preliminary laboratory experiments on the virulence of these species have shown that all of them are capable of infecting and killing local agriolimacid species collected in Moscow, of which Deroceras reticulatum is dominant. Considering that no Pellioditis species has yet been discovered in the local gastropods here in Moscow, mollusc-pathogenic nematodes new to the habitat can interact with any suitable target. It may be that native gastropods are easier hosts/victims for new nematodes. Further observations are needed to understand whether the new nematodes will be more virulent for local gastropods and how the new invaders will affect the local fauna.
Successful invaders are less affected by enemies as suggested by Colautti et al. (Reference Colautti, Ricciardi, Grigorovich and MacIsaac2004). It is possible that invasive gastropods are an exception to this rule. A number of surveys on A. vulgaris in different parts of Europe show that this species readily tolerates heavy combined nematode infection of specific gastropod parasites, with nematode loads higher than the native gastropod species (Ross et al., Reference Ross, Ivanova, Hatteland, Brurberg and Haukeland2015; Filipiak et al., Reference Filipiak, Haukeland, Zając, Lachowska-Cierlik and Hatteland2020; Antzée-Hyllseth et al., Reference Antzée-Hyllseth, Trandem, Torp and Haukeland2020). It may well be that K. melanocephalus is yet another case of this kind of tolerance to parasites.
Acknowledgements
All SEM studies were carried out at the Joint Usage Center ‘Instrumental Methods in Ecology’ at the A.N. Severtsov Institute of Ecology and Evolution.
Author contribution
E.S. Ivanova and S.E. Spiridonov contributed equally to this work. E.S. Ivanova: Conceptualization, methodology, validation, investigation, morphological identification, observation SEM, nematodes drawing, writing-original draft, writing-review & editing, visualization. S.E. Spiridonov: Sample collection, methodology, software, formal analysis, funding acquisition, validation, writing-review and editing.
Financial support
The research was supported by the Russian Science Foundation grant (19-74-20147).
Competing interests
The authors declare none.
Ethical standard
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
