CC BY
65Russian Journal of Nematology, 2019, 27 (1), 37 - 45
Preliminary data on the parasite survey of terrestrial
gastropods of Sicily
Elena Ivanova1, Mirella Clausi2, Ignazio Sparacio3 and Sergei Spiridonov1
'Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences,
Leninskii Prospect 33, 119071, Moscow, Russia 2Department of Biological, Geological and Environmental Sciences, Section of Animal Biology, University of Catania,
Via Androne 81, 95124, Catania, Italy 3via Principe di Paterno 3, 90144, Palermo, Italy e-mail: elena_s_ivanova@rambler. ru
Accepted for publication 15 August 2019
Summary. A short survey on the parasite fauna of terrestrial molluscs in south-east of Sicily was performed. In total, 432 specimens of 12 different snail species from the families Cochlostomatidae, Clausiliidae, Subulinidae, and Helicidae and five slug species from the families Agriolimacidae, Limacidae and Milacidae were collected and examined. Seven out of 17 examined gastropod species in the area were susceptible to nematode infection. Representatives of three nematode taxa, Angiostoma margaretae Ross, Malan & Ivanova, 2011, Phasmarhabditis spp. and Caenorhabditis elegans (Maupas, 1900), were isolated and molecularly characterised. New host, Papillifera papillaris affinis (Philippi, 1836), was recorded for A. margaretae, originally described from South Africa. The discovery of two different strains or even putative species of Phasmarhabditis at close localities is indicative of its possible diversity in the area. Trematodes from four families were found at four sites in Rumina decollata (Linnaeus, 1758), Cornu aspersum (O.F. Müller, 1774), Ambigolimax valentianus (Ferussac, 1822), Tandonia sowerbyi (A. Ferussac, 1823) and Milax nigricans (Philippi, 1836). Mixed trematode/nematode infection was observed in R. decollata at the University of Catania grounds in the Catania city centre, M. nigricans and T. sowerbyi at Villasmundo in Syracuse Province. No significant difference in parasite diversity and parasite prevalence between urban and wild habitats was found.
Key words: Angiostoma margaretae, Caenorhabditis elegans, morphology, morphometrics, Phasmarhabditis sp., rDNA.
Terrestrial gastropods are an important component of natural and agricultural ecosystems of the world, especially the Mediterranean region. In Sicily, particularly, geological, paleo-climatic, evolutionary and biogeographic events have contributed to a high species diversity of gastropod molluscs (Giusti & Manganelli, 1984; Manganelli et al., 1995; Bank, 2017; Bank & Neubert, 2017) with high levels of endemism (see, for example, Liberto et al., 2016; Viviano et al., 2019). Many gastropod taxa (Cochlostomatidae, Clausiliidae, Helicidae) are adapted for life in limestone areas (Colomba et al., 2011) where they can form populations of different sizes consisting of representatives of the genera Rupestrella Monterosato, 1894, Siciliaria Vest, 1867, Marmorana W. Hartmann, 1844 etc. There is also a number of invasive species in Sicily known to affect the native species diversity (McNeely, 2001; Douglas et al., 2013; Early et al., 2016; Sparacio et
al., 2017, 2018). Economically, some native as well as alien gastropod species have impact as pests in agriculture, horticulture and gardens (Manganelli et al. , 1995), while others are traditionally used as food and therefore regarded as beneficial despite occasionally being a nuisance in agriculture.
It is also known that one of the edible gastropods of Mediterranean origin, Cornu aspersum (O.F. Müller, 1774) (syn. Helix aspersa), may act as an intermediate host for a number of metastrongyloid nematodes (Seneviratna, 1959; Anderson, 2000; Gianelli et al., 2015). Such gastropod-borne parasites pose risks to health and well-being of humans and animals. Aquatic gastropods take a greater part than terrestrial ones in the distribution of trematodes, such as Schistosoma, that are dangerous for humans. However, several species of Brachylaima Dujardin, 1843, typical trematode parasite of birds, can be dangerous to humans and are transmitted by species of Monacha
© Russian Society of Nematologists, 2019; doi:10.24411/0869-6918-2019-10005
Fitzinger, 1833 (Hygromiidae) and other helicids native in the Mediterranean region (Rashed, 2008; Ismail & Gurelli, 2018). Another important nematode parasite, Angyostrongylus Kamensky, 1905, causative agent of ailments with different symptoms of domestic animals and even humans is transmitted mainly through terrestrial gastropods. Although the majority of cases of eosinophilic meningitis caused by the rat lungworm A. cantonensis (Chen, 1935) were registered outside Europe, global warming and spread of tropical gastropods around the earth by a human should be another concern in the control of major helminthiases. The wide range of gastropod hosts used by A. cantonensis shows the great adaptation potential of this parasite and points at the need for the control of its presence in gastropods. Another big group of gastropod-borne parasites is represented by nematodes (Rhabditida, Mermithida and Ascaridida) associated with gastropods as sole hosts, be it truly parasitic relationships or necromeny. Phasmarhabditis hermaphrodita (A. Schneider, 1859), a facultative (opportunistic) parasite of slugs, has been developed as the biocontrol agent sold in many countries in Europe (Wilson et al., 1993; Rae et al., 2007). The search for such natural enemies of pestilent gastropod species was recently started in Italy, and Phasmarhabditis apuliae has been described from milacid slugs (Nermut' et al., 2016). We did not find any other reports on any parasites associated with land gastropods in Italy and, to our knowledge, no studies on parasite fauna of gastropods in Sicily have been carried out. A short systematic survey on helminths of terrestrial gastropods was undertaken by the authors in Sicily in February 2019. Survey results are presented below with the focus on nematodes completing their life cycle in gastropods. Finally, some additional data on gastropod distribution in the south-east of Sicily are provided.
MATERIAL AND METHODS
Nematode material. Gastropods were collected in Sicily and transported to the Centre of Parasitology, Moscow where dissections were made. Gastropods were identified based on their morphology. Taxonomical references were based on the checklist of land and freshwater Gastropoda of Europe (Bank & Neubert, 2017). Parasites extracted were preserved for morphological and molecular studies using preservation in a hot 4-5% formalin for nematodes, 96% ethanol for trematodes for morphology and freezing for molecular work. Except for the preserved specimens, the remaining rhabditid juveniles were placed in nutrient medium (soaked in water oatmeal or preliminary frozen gastropod meat)
to obtain adult stages. Prior to molecular characterisation, the nematodes collected were identified to the generic level using light microscopy. The samples used were designated as Si5 for Angiostoma sp. from Tandonia sowerbyi (A. Ferussac, 1823) collected at Villasmundo, Phasmarhabditis sp. Si14 from Monacha sp. (Villasmundo) and Si15 from Eobania vermiculata (o.F. Müller, 1774) (Librino) and Si9 for an unidentified rhabditid from Ambigolimax valentianus (Ferussac, 1822) (University of Catania grounds).
Molecular characterisation and phylogenetic analysis. The nematodes discovered in the course of mollusc dissection were transferred individually into 25 ^l of sterile water in 0.7 ml Eppendorf tubes. DNA was extracted from worms in the worm-lysis solution (950 ^l of a mixture of 2 ml of 1M NaCl, 2 ml of 1M Tris-HCl, pH 8 plus 5.5 ml of deionised water plus 10 ^l of mercaptoethanol and 40 ^l (proteinase K, 20 mg ml-1), which was prepared immediately before DNA extraction (Holterman et al., 2006). The worm-lysis solution was incubated at 65°C for 90 min, with the following deactivation of proteinase K at 99°C during 5 min.
Primers D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') were used to amplify and sequence the D2-D3 LSU rDNA expansion segments (Nunn, 1992). The PCR cycling parameters for these two primers included a primary denaturation step at 94°C for 5 min followed by 34 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 1 min, followed by a postamplification extension step at 72°C for 6 min.
A pair of primers TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') was used to amplify approx. 800 bp long sequence of ITS region of ribosomal DNA (Curran & Driver, 1994). PCR cycling parameters included primary denaturation at 95°C for 5 min followed by 35 cycles of 94°C for 45 s, 56°C for 60 s and 72°C for 70 s. Obtained sequences were deposited in NCBI Genbank (Phasmarhabditis, Si14, ITS - MN366391 and the same species D2D3 LSU rDNA - MN366385; Phasmarhabditis, Si 15, D2-D3 LSU rDNA -MN366384). For comparative purposes and phylogeny construction, some sequences from GenBank were also used. Sequence alignments were generated using Clustal_X (Thompson et al., 1997) under default values for gap opening and gap extension penalties. All alignments were analysed using PAUP* 4.0b10 (Swofford, 1998) for maximum likelihood, maximum parsimony and neighbour joining methods.
Table 1. List of gastropod species collected in south-east Sicily in February 2019.
Location Catania
Uni. Oasi del Simeto
Species garden & Villa Bellini Villasmundo Librino Pantalica Mineo Caltagirone
Architaenioglossa
Megalostomatidae
Cochlostoma (Auritus) westerlundi dionysi (Paulucci, 1879) 0 0 0 47 0 0 0
Littorinimorpha
Bithynidae
Chondrula (Mastus) pupa (Linnaeus, 1758) 10 0 0 0 0 0 0
Styllomatophora
Achatinidae
Rumina decollata (Linnaeus, 1758) 32 7 45 0 1 8 0
Clausiliidae
Muticaria cfr. neuteboomi 0 0 0 0 9 0 0
Beckmann, 1990
Papillifera papillaris affinis (Philippi, 1836) 3 35 0 34 0 7 3
Milacidae
Milax nigricans (Philippi, 1836) 3 1 1 0 0 0 0
Tandonia sowerbyi (A. Férussac, 1823) 1 1 0 0 0 0 0
Limacidae
Ambigolimax valentianus (Férussac, 1822) 132 0 0 0 0 11 0
Limacus flavus (Linnaeus, 1758) 4 0 0 0 0 0 0
Agriolimacidae
Deroceras cfr. panormitanum (Lessona & Pollonera, 1882) 0 0 2 0 0 2 1
Trossexodontidae
Caracollina lenticula (Férussac, 1821) 0 2 0 0 0 0 0
Hygromiidae
Monacha sp. 1 1 1 0 0 0 0
Geomitridae
Cernuella (Cernuella) virgata (Da Costa, 1778) 0 0 0 0 3 0 0
Helicidae
Theba pisana (Müller, 1774) 1 0 0 2 4 6 0
Cornu aspersum (O.F. Müller, 1774) 4 1 2 0 0 0 0
Eobania vermiculata 0 1 2 0 0 0 0
(O.F. Müller, 1774)
Examined 190 49 5З S4 17 З5 4
Infected (%) З.З 20.S 17.0 0 0 5.9 0
Pellioditis sp. RS5624 KJ877242
Phasmarhabditis bonaquaense KXO17485 -Phasmarhabditis apuliae KXO 17482
18/98/97
I- Phasmarhabditis papulosa KX267674
y Phasmarhabditis sp. SA2 MF806606
i-Phasmarhabditis sp. Sil5 MN366384
491/98/95
1— Phasmarhabditis hermaphrodita KM510197
Phasmarhabditis sp. EM434 EU195967
Phasmarhabditis haizhouensis KPO17253
97/93/82
Angiostoma norvegicum R.41 MH540727 Angiostoma norvegicum KU712561
-Angiostoma norvegicum R39 MH540726
Angiostoma norvegicum R40 MH540725 Angiostoma margaretae KU712562
99/94 100
Angiostoma milacis FJ949063 Angiostoma margaretae strain SAMF192963
100/100/100
C
¡Phasmarhabditis bohémica strain CHI KXO 17484 Phasmarhabditis bohémica strain 185 KX017483
-Phasmarhabditis sp. QZL2018 MK937096
Phasmarhabditis sp. Sil4 MN366385
Phasmarhabditis meridionalis MG543920 Angiostoma limacis GQ167 725
I Phasmarhabditis hermaphrodita MK213812 loo/ioo/ioo' Phasmarhabditis hermaphrodita KM510193
Fig. 1. Phylogenetic relationships of Sicilian Phasmarhabditis strains within other nematodes based on D2-D3 LSU rDNA. Bootstrap support values are presented near nodes as MP/NJ/ML. ML analysis (500 bootstrap replications), K2 + G model. For MP and NJ - 1000 bootstrap replications.
RESULTS
A total of 432 terrestrial snails and slugs were collected from seven sample sites around Sicily in 2528 February 2019 (Table. 1). A total of 11 different snail species and five slug species were identified representing four snail families, Cochlostomatidae, Clausiliidae, Helicidae and Subulinidae, and three slug families, Agriolimacidae, Limacidae and Milacidae. Trematodes were found at four sites and were represented by four families. Mollusc species infected by trematodes were Rumina decollata (Linnaeus, 1758), Cornu aspersum, Ambigolimax valentianus, Tandonia sowerbyi and Milax nigricans (Philippi, 1836). Mixed trematode/nematode infection was observed in R. decollata collected at University of Catania grounds in the centre of Catania, M. nigricans and T. sowerbyi at Villasmundo Natural Reserve in Syracuse Province.
Molecular characterisation. The sequences of D2-D3 expansion segment were obtained for four
samples (Si5, Si9, Si 14 and Si 15) of nematodes from Sicilian molluscs. The approx. 500 bp long D2-D3 LSU rDNA sequence for the sample Si5 Angiostoma sp. was found to be identical with South African Angiostoma margaretae Ross, Malan & Ivanova, 2011 (deposited sequences KU712562 and MF192963 of specimens isolated from Milax gagates Draparnaud, 1801 and Deroceras panormitanum (Lessona & Pollonera, 1882), respectively). A BLAST-search demonstrated that the deposited ITS sequence MF 192968 of A. margaretae from South African slug Deroceras panormitanum was the closest to the 730 bp long ITS rDNA sequence of our sample.
The 590 bp long sequence for sample Si9 was found to be 100% identical to the LSU sequence of the nematode Caenorhabditis elegans isolated in South Africa from the cosmopolitan slug Deroceras panormitanum (MF 192964) and C. elegans from Asian horned frogs Megophrys montana (FJ589010). The 940 bp long ITS rDNA sequence was obtained for this sample. This ITS sequence of Si9
100/
-Ancylostoma can inn m KC755029
-Phasmarhabditis sp. QZL2018 MK542667
00/1001 Phasmarhabditis bohémica KX017490 I-Phasmarhabditis bohémica KX017489
100/100/1 93/98/95 100; £>7/
99/94/100
100
100/100/1
fiöi
10/88/96
10
100/93/100
DO! Phasmarhabditis papillosa KM510205
Phasmarhabditis papillosa KX267675
Phasmarhabditis apuliae KX017486
Phasmarhabditis apuliae KX017487
Phasmarhabditis apuliae KX017488
Phasmarhabditis californica KM510204
Phasmarhabditis californica KM510203 Phasmarhabditis sp. Sil4 MN366391
— Phasmarhabditis neopapillosa FJ516760 Phasmarhabditis hermaphrodita MK214817 Phasmarhabditis hermaphrodita KM510202 Phasmarhabditis hermaphrodita FJ516761 Phasmarhabditis hermaphrodita MG551718 Phasmarhabditis hermaphrodita KM510201
Fig. 2. Phylogenetic relationships of Sicilian Phasmarhabditis strains within other nematodes based on ITS rDNA. Bootstrap support values are presented near nodes as MP/NJ/ML. ML analysis (500 bootstrap replications), T92 + G model. For MP and NJ - 1000 bootstrap replications.
demonstrated complete identity in 820 corresponding positions of ITS sequence for C. elegans from an unidentified European slug (MG551716).
The D2-D3 LSU rDNA sequences obtained for samples Si14 and Si15 as expected corresponded to members of Phasmarhabditis. The 540 bp long sequence of Si14 clustered in the phylogenetic tree (Fig. 1) with a group of Phasmarhabditis species, which included P. bohemica Nermut', Puza, Mekete & Mracek, 2017, P. hermaphrodita (Schneider, 1859) Andrassy, 1983, P. meridionalis Ivanova & Spiridonov, 2017, and an unidentified Phasmarhabditis sp. QZL2018 from China (MK937096), and also Angiostoma limacis Dujardin, 1845 GQ167725. The 649 bp long ITS rDNA sequence of Si14 demonstrated another phyletic links. It clustered with P hermaphrodita and P. neopapillosa (Mengert in Osche, 1952)
Andrassy, 1983 ITS sequences under strong bootstrap support (Fig. 2).
The 496 bp long D2-D3 LSU rDNA sequence of Si15 was quite different from sample Si14 clustering with P hermaphrodita (KM510197) from Deroceras reticulatum slug collected in the USA.
Characterisation of collection sites, their gastropod population and associated parasites. I. Catania, the University garden and Villa Bellini city park. At Villa Bellini, we were able to find just a few gastropod specimens (Milax nigricans, Tandonia sowerbyi and Cernuella virgata), all free of the helminth's infection. At the same time, the University garden, a small area around the Section of Animal Biology, was inhabited by large numbers of gastropods, which sought shelter under lids of technical appliances (hatches and manhole covers). There, the gastropod population was represented by two slug species (Limacus flavus and Ambigolimax
valentianus) and five snail species (Rumina decollata, Chondrula (Mastus) pupa, Monacha sp., Cornu aspersum, Cernuella virgata and Papillifera papillaris affmis). Predominant species were A. valentianus and R. decollata, which accounted to 72.9% and 17.7% of sampled gastropods, respectively. Total prevalence of helminth infection at the University grounds was 6% (A. valentianus, R. decollata and C. aspersum). Two species of nematodes and a species of a trematode were recorded: unidentified Phasmarhabditis sp. (C. aspersum and R. decollata); Caenorhabditis elegans (A. valentianus), and Dicrocoeliidae gen. et sp. (R. decollata). The infected specimen of R. decollata has served as a host for two parasites at the same time: a nematode Phasmarhabditis sp. and trematode sporocysts of Dicrocoeliidae, a parasite of mammals and birds with a complicated life cycle. To the best of our knowledge, this gastropod species was not reported previously as the intermediate host for Dicrocoeliidae.
While the juveniles of Phasmarhabditis sp. inhabited the mantle cavities of C. aspersum and R. decollatum and accounted from 3 to 10 specimens, hundreds of juveniles of C. elegans were found in the intestine of one out of 132 collected A. valentianus. The C. elegans dauer juveniles were tightly packed in distal parts of the intestine and were alive, while obviously getting ready for evacuation. What was the source of infection for slugs and why only one slug specimen was infected remained unclear. In the lab, the nematodes were able to multiply on oatmeal medium and on the frozen Helix meat with better results on the latter.
II. Librino. Librino is the suburb district of Catania, located in the south-west part of the town. The gastropods were collected in an uncultivated green area, on the edge of the town (slugs Milax nigricans, Deroceras cfr. panormitanum and snails R. decollata, C. aspersum, E. vermiculata and Monacha sp.). Here, R. decollata were predominant and 17.8% were found infected with Phasmarhabditis sp. Si 15. The same strain was recovered from the only specimen of E. vermiculata collected nearby. One of two specimens of C. aspersum hosted metacercariae of Brachylaimidae. Total prevalence was 16.9%.
III. Highway 417 by Mineo. Examination of the small collection of snails (Cernuella virgata, Papillifera papillaris affinis, Muticaria cfr. neuteboomi, Theba pisana, R. decollata), collected at limy slopes by Mineo did not reveal parasites.
IV. The Natural Reserve "Oasi del Simeto". Oasi del Simeto is a wetland with the pioneer vegetation of dunes at the mouth of Simeto river, south to Catania, providing a home for numerous
birds. The site hosted a small population of gastropods, represented by both snails and slugs (Deroceras cfr. panormitanum, A. valentianus, R. decollata, C. virgata and Theba pisana). Total prevalence was 5.9%: a specimen of A. valentianus was infected by metacercariae of Brachylaimidae and R. decollata by Phasmarhabditis sp. (no molecular characteristics yet).
V. The Natural Reserve "Speleological Complex Villasmundo - S. Alfio". The sampling site in the Villasmundo Reserve in Syracuse Province was located on the mountain slope with the steppe vegetation with rare carob and olive trees and hawthorns. Here, representatives of eight gastropod species were collected (Milax nigricans, Tandonia sowerbyi, Papillifera papillaris affinis, R. decollata, E. vermiculata, C. aspersum, Caracollina lenticular and Monacha sp.). Their examination has shown a surprisingly high diversity of its helminths (two species of nematodes and two species of trematodes) together with the highest prevalence in the survey (20.8%). Gastropod species infected at this site were M. nigricans, T. sowerbyi, P. papillaris affinis, E. vermiculata and Monacha sp. Phasmarhabditis sp. Si 14 was found in the representatives of each gastropod species. The intestinal parasite Angiostoma margaretae (Si5) was found in T. sowerbyi and P papillaris affinis. This nematode (1 to 6 specimens) was recovered from the oesophagus of its gastropod host and was represented by adult forms as well as late juveniles.
Both slug species examined (one specimen of each species was collected) showed cases of co-infection: in M. nigricans, it was Phasmarhabditis sp. + sporocysts of trematodes and in T. sowerbyi, metacercariae of Brachylaimidae + Phasmarhabditis sp. and Angiostoma margaretae.
VI. The Pantalica Nature Reserve. The sampling site at Pantalica necropolis was located close to Sortino (Syracuse Province) at 328 m a.s.l. and characterised mostly by vegetation of Mediterranean scrub. The following gastropod species were found there: Cochlostoma (Auritus) westerlundi dionysi (predominant), P papillaris affinis and T. pisana. None was infected.
VII. Caltagirone. Several specimens of P. papillaris affinis and Deroceras cfr. panormitanum were collected in the city park and the latter was infected by an unidentified Phasmarhabditis sp.
DISCUSSION
Our survey covered just a minor portion of terrestrial gastropod diversity of the south-east part of Sicily. In general, the gastropods collected were
represented by species with a wide distribution range and the majority of gastropods examined during our study were snails. No significant difference in the parasite prevalence between them was found and both slugs and snails were found associated with parasites in our study (three species of slugs and four of snails). Slugs are considered to be more often affected by nematodes due to their better exposure to the soil (Rae, 2018). There is probability that in Sicily and similar habitats snail species may play the greater role as nematode hosts in the area due to their predominance over slugs.
Examination of more than four hundred land gastropods in Sicily showed quite low infestation rate typical for gastropod populations in Europe (Ivanova et al., 2013; Ross et al., 2015; Singh et al., 2019) and USA (Ross et al., 2010), while considering that previous surveys were focused only on slugs and their nematode parasites. No significant difference in parasite diversity and parasite prevalence between urban and wild habitats was found (highest values 17% at Librino and 20.8% at Villasmundo and lowest 3.3% at Catania University grounds and 0% Pantalica). Moreover, in the two well represented sites, the urban (Librino) and the wild (Villasmundo), parasite prevalence values were similar despite different gastropod species being present. However, more collections should be made and analysed to obtain a large body of data to allow further conclusions.
In our study, some of collected gastropod species were found in small quantities, thus preventing speculations on its susceptibility to parasites. Conversely, the only non-pulmonate snail species, Cochlostoma (Auritus) westerlundi dionysi, was found in quite large numbers and all snails were free of infection. To determine if the species is resistant to nematodes, examination from another localities should be made.
However, even the small scale of the survey demonstrated that many gastropod species (at least seven out of 17 examined) in the area were susceptible to nematode infection. Representatives of three taxa (Angiostoma margaretae, Phasmarhabditis spp. and C. elegans) were molecularly characterised, while yet another rhabditid species recovered and possible Alloionema sp. are in need of further collection and examination. All nematodes mentioned were recovered from living gastropod specimens. The discovery of two different strains or even putative species of Phasmarhabditis at close localities points at its possible variety in the area. A new host (Papillifera papillaris affnis) was recorded for A. margaretae. The nematode was found in Villasmundo in two non-related gastropod hosts: Papillifera papillaris affinis
(Clausiliidae) and Tandonia sowerbyi (Milacidae). It was originally described from a cosmopolitan milacid slug Milax gagates in South Africa (Ross et al., 2011). Later, it was found in several slug species native for South Africa (own data, unpubl.). How many gastropod species inhabiting the same site were utilised by this parasite has yet to be investigated.
Petersen et al. (2015) reported that C. elegans was found in slugs quite often (9.9%) in the course of their survey on invertebrate vectors of C. elegans in northern Germany and slugs' intestines were the common habitat. In that study, the gastropod host range of C. elegans included mostly slugs of the Arionidae family, whereas no arionid slugs were collected during our survey. These authors tested the ability of C. elegans from an intestine to proliferate and proved it possible. They suggested that slug's intestine provides the suitable (humid and microberich) environment for C. elegans and discussed the nature of relationships between C. elegans and gastropods (phoretic, commensal or even parasitic?). In our study, the behaviour of C. elegans rather supports the possible phoretic nature of the relationship between the nematode and the gastropod considering the huge amount of C. elegans juveniles (hundreds) carried in A. valentianus intestines.
In our study, we did not use the specific (histopathology) methods for the discovery of tissue-dwelling nematode juveniles living in a gastropod as an intermediate host. However, it is known that juveniles of Metastrongyloidea are being shed from the foot where they accumulate in the environment after reaching the invasive stage (Gianelli et al., 2015). In our study, the examination of the mucus of all gastropods collected did not reveal the presence of metastrongylid juveniles.
ACKNOWLEDGEMENTS
This work was conducted under the IEE RAS Government basic research program (project no. 0109-2018-0075). The research was partially funded by Annual Research Plan 2016-18 of Dept. Biological Geological Environmental Sciences, University of Catania (grant no. 22722132131). The financial support of a grant 17-04-00095a from the Russian Fund for Basic Research is appreciated.
REFERENCES
Anderson, R.C. 2000. Nematode Parasites of
Vertebrates: Their Development and Transmission.
UK, CAB International. 650 pp.
DOI: 10.1079/9780851994215.0000
AndräSSY, I. 1983. A taxonomic review of the suborder Rhabditina (Nematoda: Secernentea). France, O.R.S.T.O.M. 241 pp. Bank, R.A. 2017. Fauna Europaea Project. Checklist of the
Land and Freshwater Gastropoda of Europe. 176 pp. Bank, R.A. & Neubert, E. 2017. MolluscaBase. Checklist of the Land and Freshwater Gastropoda of Europe. 170 pp. URL: https://www.molluscabase.org (accessed: August 10, 2019). Colomba, M.S., Gregorini, A., Liberto, F., Reitano, A., Giglio, S. & Sparacio, I. 2011. Monographic revision of the endemic Helix mazzullii De Cristofori & Jan, 1832 complex from Sicily and re-introduction of the genus Erctella Monterosato, 1894 (Pulmonata, Stylommatophora, Helicidae). Zootaxa 3134: 1-42. Curran, J. & Driver, F. 1994. Molecular taxonomy of Heterorhabditis. In: COST 812. Biotechnology. Genetics of Entomopathogenic Nematode - Bacterium Complexes. Proceedings of a Symposium and Workshop held at St. Patrick's College, Maynooth, Co. Kildare, Ireland and National reports 1990-1993 (A.M. Burnell, R-U. Ehlers & J.P. Masson Eds). pp. 41-48. Brussels, Belgium, office for official Publications of the European Communities. Douglas, D.D., Brown, D.R. & Pederson, N. 2013. Land snail diversity can reflect degrees of anthropogenic disturbance. Ecosphere 4: 28. DOI: 10.1890/ES12-00361.1 Early, R., Bradley, B.A., Dukes, J.S., Lawler, J.J., Olden, J.D., Blumenthal, D.M., Gonzalez, P., Grosholz, E.D., Ibanez, I., Miller, L.P., Sorte, C.J.B. & Tatem, A.J. 2016. Global threats from invasive alien species in the twenty-first century and national response capacities. Nature Communications 7: 12485. DOI: 10.1038/ncomms12485 Giannelli, A., Colella, V., Abramo, F., do Nascimento Ramos, R.A., Falsone, L., Brianti, E., Varcasia, A, Dantas-Torres, F., Knaus, M, Fox, M.T. & Otranto, D. 2015. Release of lungworm larvae from snails in the environment: potential for alternative transmission pathways. PLoS Neglected Tropical Diseases 9: e0003722.
DOI: 10.1371/journal.pntd.0003722 Giusti, F. & Manganelli, G. 1984. Relationships between geological land evolution and present distribution of terrestrial gastropods in the western Mediterranean area. In: World-Wide Snails. Biogeographical Studies on Non-Marine Mollusca (A. Solem & A.C. van Bruggen Eds). pp. 70-90. Leiden, The Netherlands, Brill. Holterman, M., Van der Wurff , A., Van den Elsen, S., Van Megen, H., Bongers, T., Holovachov, O., Bakker, J. & Helder, J. 2006. Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution towards
crown clades. Molecular Biology and Evolution 23: 1792-1800. DOI: 10.1093/molbev/msl044 Ismail, F.M.A.K. & Gürelli, G. 2018. Natural infection of the land snails Xerolenta obvia, Zebrina detrita, and Monacha samsunensis by Brachylaima sp. metacercariae in Kastamonu, Turkey. Turkish Journal of Zoology 42: 152-157. DOI: 10.3906/zoo-1705-61 Ivanova, E.S. & Spiridonov, S.E. 2017. Phasmarhabditis meridionalis sp. n. (Nematoda: Rhabditidae) from a land snail Quantula striata (Gastropoda: Dyakiidae) from south of Vietnam. Russian Journal of Nematology 25: 129-140. Ivanova, E.S., Panayotova-Pencheva, M.S. & Spiridonov, S.E. 2013. Observations on the nematode fauna of terrestrial molluscs of the Sofia area (Bulgaria) and the Crimea peninsula (Ukraine). Russian Journal of Nematology 21: 41-49. Liberto, F., Reitano, A., Giglio, S., Colomba, M.S. & Sparacio, I. 2016. Two new Clausiliidae (Gastropoda Pulmonata) of Sicily (Italy). Biodiversity Journal 7: 365-384.
Manganelli, G., Bodon, M., Favilli, L. & Glusti, F. 1995. Gastropoda Pulmonata. In: Checklist delle Specie della Fauna Italiana, Volume 16 (A. Minelli, S. Ruffo & S. La Posta Eds). pp. 1-60. Bologna, Italy, Universita di Bologna. McNeely, J. 2001. Invasive species: a costly catastrophe for native biodiversity. Land Use and Water Resources Research 1: 1-10. Nermut', J., Püza, V. & Mräcek, Z. 2016. Phasmarhabditis apuliae n. sp. (Nematoda: Rhabditidae), a new rhabditid nematode from milacid slugs. Nematology 18: 1095-1112. DOI: 10.1163/15685411-00003017 Nermut', J., Püza, V., Mekete, T. & Mräcek, Z. 2017. Phasmarhabditis bohemica n. sp. (Nematoda: Rhabditidae), a slug-parasitic nematode from the Czech Republic. Nematology 19: 93-107. DOI: 10.1163/15685411-00003034 Nunn, G.B. 1992. Nematode molecular evolution. Ph.D. Dissertation, University of Nottingham, Nottingham, UK, 187 pp.
Petersen, C., Hermann, R.J., Barg, M.C., Schalkowski, R., Dirksen, P., Barbosa, C. & Schulenburg, H. 2015. Travelling at a slug's pace: possible invertebrate vectors of Caenorhabditis nematodes. BMC Ecology 15: 19. DOI: 10.1186/s12898-015-0050-z Rae, R. 2018. Shell encapsulation of parasitic nematodes by Arianta arbustorum (Linnaeus, 1758) in the laboratory and in field collections. Journal of Molluscan Studies 84: 92-95. DOI: 10.1093/mollus/eyx045 Rae, R., Verdun, C., Grewal, P., Robertson, J.F. & Wilson, M.J. 2007. Biological control of terrestrial
molluscs using Phasmarhabditis hermaphrodita -progress and prospects. Pest Management Science 63: 1153-1164. DOI: 10.1002/ps.1424 Rashed, A.A. 2008. A new parasitic metacercaria from the land snail Monacha obstructa Pfeiffer, 1842 with critical review on relevant metacercariae belonging to the genus Brachylaima Dujardin, 1843. Journal of Egyptian Society of Parasitology 38: 483-500. Ross, J.L., Malan, A.P. & Ivanova, E.S. 2011. Angiostoma margaretae n. sp (Nematoda: Angiostomatidae), a parasite of the milacid slug Milax gagates Draparnaud collected near Caledon, South Africa. Systematic Parasitology 79: 71-76. DOI: 10.1007/s11230-011-9294-y Ross, J.L., Ivanova, E.S., Severns, P.M. & Wilson, M.J. 2010. The role of parasite release in invasion of the USA by European slugs. Biological Invasions 12: 603-610. DOI: 10.1007/s10530-009-9467-7 Ross, J.L., Ivanova, E.S., Hatteland, B.A., Brurberg, M.B. & Haukeland, S. 2015. Survey of nematodes associated with terrestrial slugs in Norway. Journal of Helminthology 5: 583-587. DOI: 10.1017/S0022149X15000784 Seneviratna, P. 1959. Studies on Anafilaroides rostratus Gerichter, 1949 in cats. The Adult and its first stage larva. Journal of Helminthology 33: 99-108. Singh, P.R., Couvreur, M., Decraemer, W. & Bert, W. 2019. survey of slug parasitic nematodes in East and West Flanders, Belgium and description of Angiostoma gandavensis n. sp. (Nematoda: Angiostomidae) from arionid slugs. Journal of Helminthology 93: 1-11. DOI: 10.1017/S0022149X19000105
Sparacio, I., La Mantia, T., Reitano, A., Colomba, M.S. & Liberto, F. 2018. Nuovi dati biologici e geonemici su Lehmania melitensis (Lessona & Pollonera, 1882) e Ambigolimax valentianus (A. Ferussac, 1823) (Gastropoda: Limacidae) in Sardegna, Sicilia (Italia) a Gozo (Isole Maltesi). Alleryana 36: 99-106. Sparacio, I., La Mantia, T., Colomba, M.S., Liberto, F., Reitano, A. & Giglio, S. 2017. Qanat, gebbie and water sources: the last refuge for the malacological freshwater fauna in Palermo (Sicily, Italy). Biodiversity Journal 8: 279-310. Swofford, D.L. 1998. PAUP*. Phylogenetic Analysis Using Parsimony (* and Other Methods). Version 4. USA, Sinauer Associates, Inc. 128 pp. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. 1997. The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24: 4876-4882. DOI: 10.1093/nar/25.24.4876 Viviano, R., Viviano, A, Liberto, F., Reitano, A. & Sparacio, I. 2019. A new species of the genus Schileykiella Manganelli, Sparacio et Giusti, 1989 from Sicily (Italy) (Gastropoda Pulmonata Canariellidae). Biodiversity Journal 10: 71-80. DOI: 10.31396 Wilson, M.J., Glen, D.M. & George, S.K. 1993. The rhabditid nematode Phasmarhabditis hermaphrodita as a potential biological control agent for slugs. Biocontrol Science and Technology 3: 503-511. DOI: 10.1080/09583159309355306.
E.A. Ivanova, M. Clausi, I. Sparacio and S.E. Spiridonov. Предварительные результаты обследования паразитов наземных гастропод Сицилии.
Резюме. Проведено обследование паразитофауны наземных гастропод юго-востока Сицилии. Было собрано и обследовано 432 моллюска, принадлежащих к 12 видам улиток из семейств Cochlostomatidae, Clausiliidae, Subulinidae и Helicidae и 5 видам слизней из семейств Agriolimacidae, Limacidae и Milacidae. Инвазия нематод была обнаружена у 7 из 17 обследованных видов гастропод. Представители трех таксонов нематод (Angiostoma margaretae Ross, Malan & Ivanova, 2011, Phasmarhabditis spp. и Caenorhabditis elegans (Maupas, 1900) были выделены и охарактеризованы молекулярными методами. Для A. margaretae, первоначально описанной из Южной Африки, был зарегистрирован новый хозяин - Papillifera papillaris affinis (Philippi, 1836). Обнаружены два штамма (или, возможно, вида) Phasmarhabditis, что указывает на высокую вероятность их разнообразия в регионе. Трематоды были представлены 4 семействами и обнаружены в 4 из 7 мест сбора у Rumina decollata (Linnaeus, 1758), Cornu aspersum (O.F. Müller, 1774), Ambigolimax valentianus (Férussac, 1822), Tandonia sowerbyi (A. Férussac, 1823) и Milax nigricans (Philippi, 1836). Смешанная нематодно-трематодная инвазия была обнаружена у R. decollata на территории Университета Катании, а также у M. nigricans и T. sowerbyi в Вилласмундо, провинция Сиракузы. Не обнаружено существенной разницы в индексах разнообразия и распространенности паразитов между городскими и дикими популяциями гастропод.
