Introduction
Current understanding of nematodes associated with terrestrial slugs is based on surveys conducted in Germany (Mengert, Reference Mengert1953), France (Morand, Reference Morand1988), Slovenia (Laznik et al., Reference Laznik, Ross, Tóth, Lakatos, Vidrih and Trdan2009), the Crimea (Ivanova et al., Reference Ivanova, Panayotova-Pencheva and Spiridonov2013), Bulgaria (Ivanova et al., Reference Ivanova, Panayotova-Pencheva and Spiridonov2013), the USA (Gleich et al., Reference Gleich, Gilbert and Kutscha1977; Ross et al., Reference Ross, Ivanova, Severns and Wilson2010a, b), Australia (Charwat & Davies, Reference Charwat and Davies1999), Africa (Ross et al., Reference Ross, Ivanova and Malan2011, Andersen et al., Reference Andersen, Haukeland, Hatteland and Westrum2012) and the UK (Ross et al., Reference Ross, Ivanova, Severns and Wilson2010a, b). A total of seven nematode families have been found to parasitize terrestrial slugs, including Agfidae, Alloionematidae, Angiostomatidae, Cosmocercidae, Diplogasteridae, Mermithidae and Rhabditidae; however, Phasmarhabditis hermaphrodita (Rhabditidae) is the only nematode to have been developed commercially as a biological molluscicide (Rae et al., Reference Rae, Verdun, Grewal, Robertson and Wilson2007). Phasmarhabditis hermaphrodita is pathogenic to a range of slug families, including Agriolimacidae, Arionidae, Limacidae, Milacidae and Vagnulidae (Rae et al., Reference Rae, Verdun, Grewal, Robertson and Wilson2007). The infectious nematode larvae seek out slugs and enter through natural openings. Once inside, slug feeding is inhibited, with death usually following 4–21 days after nematode infection (Tan & Grewal, Reference Tan and Grewal2001).
The invasive slug Arion vulgaris (also regarded as Arion lusitanicus Mabille, 1968) originates from south-western Europe, and was first reported in Norway in 1988 (Von Proschwitz & Winge, Reference Von Proschwitz and Winge1994). Since then, it has become a major pest in ornamental and vegetable plots, as well as being a serious nuisance pest (Hofsvang et al., Reference Hofsvang, Haukeland and Andersen2008). Of major concern is the spread of A. vulgaris to cultivated agricultural and horticultural crops such as strawberries and vegetables (Andersen et al., Reference Andersen, Haukeland, Hatteland and Westrum2012). Once established, populations of A. vulgaris are difficult to manage, especially in years of favourable slug weather, where mild winters are followed by a wet spring and summer. The reasons for successful colonization of A. vulgaris are not fully understood; however, typical for invasive organisms is their flexible behaviour that allows for relatively easy adaption to a new environment. Ross et al. Reference Ross, Ivanova, Severns and Wilson(2010a) have also demonstrated that parasite release plays an important role in successful mollusc invasions. This paper presents new data on the diversity and distribution of nematode parasites that are associated with terrestrial slugs in Norway.
Materials and methods
Collection and identification of slugs
In the current study, adult slugs were collected from suitable localities that were found in cooperation with local gardeners, growers and agricultural advisory services. Coordinates and land use were recorded. Slugs were collected during late August, September and October 2011. Slugs were identified through morphological examination and dissection of their genitalia (Hatteland et al., Reference Hatteland, Solhøy, Schander, Skage, Von Proschwitz and Noble2015; Von Proschwitz, pers. comm.). For slugs identified as A. vulgaris and Arion ater, the mitochondrial DNA was analysed (Folmer et al., Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994; Hatteland et al., Reference Hatteland, Solhøy, Schander, Skage, Von Proschwitz and Noble2015).
Nematode identification
The slugs were dissected and examined for nematode parasites. Nematodes were identified using a combination of morphological and molecular techniques. Morphological identification was conducted using a Zeiss Jenaval microscope (Zeiss, Germany). The adult nematodes, after being heat killed at 60°C in 5% formaldehyde, were processed using anhydrous glycerol for purposes of mounting and identification (Ross et al., Reference Ross, Ivanova, Sirgel, Malan and Wilson2012). To obtain the adult stages of rhabditid juveniles for morphological identification, juvenile bacterial-feeding nematodes were cultured on modified kidney medium (Ross et al., Reference Ross, Ivanova, Sirgel, Malan and Wilson2012), pre-inoculated with the bacterium Moraxella osloensis. This method is known to be effective for rearing Phasmarhabditis spp. (Ross et al., Reference Ross, Ivanova, Sirgel, Malan and Wilson2012). For purposes of molecular identification, the nematodes, after storage in 70% ethanol, were transferred to Chelex/Proteinase K mix to allow for DNA extraction (Ross et al., Reference Ross, Ivanova, Spiridonov, Waeyenberge, Moens, Nicol and Wilson2010b). Characterization of the partial 18S rRNA gene was then conducted, using the methods described by Ross et al. Reference Ross, Ivanova, Spiridonov, Waeyenberge, Moens, Nicol and Wilson(2010b). Raw sequences were trimmed and assembled using Sequencher 4.1 (Genes Codes Corp., Ann Arbor, Michigan, USA). Sequences were used for searching GenBank with the BLASTn search tool (Altschul et al., Reference Altschul, Gish, Miller, Myers and Lipman1990).
Results
In total, 611 terrestrial slugs were collected from 32 sample sites around Norway. Twelve slug species were identified, representing four different slug families (table 1). Nematodes were found to have parasitized seven of the 12 slug species identified (table 1). Slugs that were free from nematode parasites included Deroceras panormitanum, Arion circumscriptus, Arion distinctus, Limax cinereoniger and Milax gagates (table 1). The two most abundant slug species collected in Norway were Deroceras reticulatum (260 slugs) and A. vulgaris (204 slugs). Of these, 3.1% of D. reticulatum were found to be infected with nematodes, compared to 34.8% of A. vulgaris (table 1).
Table 1 The prevalence (%) and intensity of infection of terrestrial slugs with nematode species from Norway; N, number of slugs examined.
bm, buccal mass; cp, crop; fs, foot sole; in, intestine; mc, mantle cavity; oe, oesophagus; rs, reproductive system; sg, salivary glands; sp, spermatheca.
* New host association.
† First record in Norway.
Nematodes were found to be parasitizing slugs at 20 of the 32 sites examined, i.e. 62.5% of the sample sites used. Of all the slugs, 18.7% were found to be infected with nematodes. A total of five nematode species were identified using morphological characteristics and 18S sequencing. As sequences of the same species were found to be identical, only one representative sequence was submitted for each taxon involved (table 2). The identified species were Alloionema appendiculatum Schneider, 1859; Agfa flexilis Dujardin, 1845; Angiostoma limacis Dujardin, 1845; Angiostoma sp., and Phasmarhabditis hermaphrodita (Schneider, 1859) Andrássy, 1983. Of the 20 positive sample sites, nine sites were found to be infected by A. appendiculatum, three sites by A. flexilis, four sites by A. limacis, seven sites by Angiostoma sp. and 14 sites by P. hermaphrodita. The most heavily infected sites were Avaldsnes (59°17′29″N, 05°13′39″E), Haukedalen (60°27′49″N, 05°17′58″E), Leirvik (59°46′24″N, 05°29′44″E), Melsum Vik (59°15′04″N, 10°21′13″E) and Utne (60°55′54″N, 06°62′47″E). Of all species collected, only one nematode was previously undescribed. (Angiostoma sp.). This is the first record of A. appendiculatum, A. flexilis and A. limacis in Norway.
Table 2 The partial 18S rRNA gene accession numbers of five nematode species from slugs in Norway with NCBI matches (EU573704, EU573707, EU573705, EU196008, DQ639980); ranges in identity and coverage are 97–100%.
Discussion
This paper presents the findings of a new survey on the diversity and distribution of slug-parasitic nematodes in Norway. Five nematode species were identified in Norway – A. appendiculatum Schneider, 1859, A. flexilis Dujardin, 1845, A. limacis Dujardin, 1845, Angiostoma sp. and P. hermaphrodita (Schneider, 1859) Andrássy, 1983. These nematodes represent four different families: Alloionematidae, Agfidae, Angiostomatidae and Rhabditidae.
Alloionema appendiculatum was found at nine of the 32 sample sites examined, and is the first record in Norway. The nematode was found to parasitize the foot sole of arionids (A. ater, A. fuscus, A. rufus, A. vulgaris), and this is the first record of its association with an A. rufus/A. ater hybrid. The family Alloionematidae is known from two genera, Rhabitophanes and Alloionema. Rhabitophanes commonly associates with insects, while the monotypic genus, Alloionema, is represented by the mollusc-parasitic nematode, A. appendiculatum (Schuurmans-Stekhoven, Reference Schuurmans-Stekhoven1950). Charwat & Davies (Reference Charwat and Davies1999) demonstrated that A. appendiculatum is non-pathogenic to mollusc hosts, while more recent work illustrates that the nematode is capable of significant mortality in A. lusitanicus (Laznik et al., Reference Laznik, Ross, Tóth, Lakatos, Vidrih and Trdan2009). Cabaret & Morand (Reference Cabaret and Morand1990) also suggested that the nematode is pathogenic to several snail species.
In the current study, A. flexilis associated with A. vulgaris and Limax maximus, and was found to be present at three of the 32 sample sites. This is the first record of the presence of A. flexilis in Norway, as well as being the first time that it has been associated with A. vulgaris. Three species of the family Agfidae are known: A. flexilis, Agfa morandi and Agfa tauricus (Morand et al., Reference Morand, Wilson, Glen and Barker2004). Agfa morandi is known by a single record in the French Pyrenees, while A. tauricus was found in several localities in the Crimea and Bulgaria (Ivanova et al., Reference Ivanova, Panayotova-Pencheva and Spiridonov2013). However, A. flexilis has been found further afield in Europe, the USA and Africa (Morand et al., Reference Morand, Wilson, Glen and Barker2004; Ross et al., Reference Ross, Ivanova, Severns and Wilson2010a, Reference Ross, Ivanova, Spiridonov, Waeyenberge, Moens, Nicol and Wilsonb, Reference Ross, Ivanova, Sirgel, Malan and Wilson2012). All three species parasitize limacid hosts; however, A. tauricus has also been recorded from agriolimacid slugs and a zonitid snail. All three nematodes are obligate parasites of molluscs and little is known about their life cycles, apart from their association with the salivary gland or genital tract (Morand et al., Reference Morand, Wilson, Glen and Barker2004), albeit the localization of the nematodes in the reproductive tract of a mollusc has been subject to dispute (Ivanova et al., Reference Ivanova, Panayotova-Pencheva and Spiridonov2013).
Two angiostomatids, A. limacis and Angiostoma sp., were identified. This is the first record of the presence of A. limacis in Norway. Generally, its distribution is based on the presence of hosts from the Arionidae, and, rarely, the Agriolimacidae, family. The latter, representing the new species, is currently undergoing description. However, it is known to closely resemble A. limacis morphologically. The Angiostomatidae family has two known genera, Angiostoma with 17 species, and Aulacnema, which is monotypic. The latter genus is a parasite of a tropical terrestrial snail. Molluscan angiostomatids are generally obligate parasites of the intestine, hepato-pancreas (Ivanova & Spiridonov, Reference Ivanova and Spiridonov2010) and oesophagus (Ross et al., Reference Ross, Ivanova and Malan2011). Currently, 13 angiostomatids have been described as possessing mollusc hosts, with eight of the former having been reported from Europe and the UK. The remaining four species have been isolated from the intestine and bronchi of amphibian and reptile hosts (Falcon-Ordaz et al., Reference Falcon-Ordaz, Mendoza-Garfias, Windfield-Perez, Parra-Olea and De Leon2008). Falcon-Ordaz et al. (Reference Falcon-Ordaz, Mendoza-Garfias, Windfield-Perez, Parra-Olea and De Leon2008) suggest that the salamander parasite Angiostoma lamotheargumedoi could have resulted from the host-switching event of a gastropod. Grewal et al. (Reference Grewal, Grewal, Tan and Adams2003) define angiostomatids as being parasites of vertebrates, using molluscs as obligate intermediate hosts. However, no angiostomatid species has yet been reported as being from both an invertebrate (supposedly intermediate) and a vertebrate (definitive) host. In both kinds of hosts, parasites have presented in their adult stages, thus showing the finalization of their development in a single host. A clear indication of the presence of separate, directly transmitted parasites has been found (Poulin, Reference Poulin2011), leading to the need for each case of invasion to be regarded separately. To date, the molecular affiliations of the nematode species from vertebrates are still unknown, thus preventing the resolution of the relationships between the two parts of the genus.
Phasmarhabditis hermaphrodita (Rhabditidae family) was also identified in the survey. Several genera within the Rhabditidae family, including Rhabditis, Caenorhabditis and Phasmarhabditis, are known to associate with slugs. However, Phasmarhabditis is the only genus that is considered to be truly parasitic towards slugs (Morand et al., Reference Morand, Wilson, Glen and Barker2004). Phasmarhabditis spp. parasitize the mantle cavity of slugs, in close association with the shell, where present (in Limacoidea slugs). However, unlike the obligate parasites, Agfa and Angiostoma, Phasmarhabditis spp. are facultative parasites that are known to live on leaf litter and slug faeces (Tan & Grewal, Reference Tan and Grewal2001; MacMillan et al., Reference MacMillan, Haukeland, Rae, Young, Crawford, Hapca and Wilson2009). Phasmarhabditis spp. usually display a remarkably high level of pathogenicity against their hosts; however, for A. vulgaris, the nematode can only control slugs under the weight of 1 g, indicating that the nematode is only effective against juvenile slug stages (Speiser et al., 2001; Grimm, Reference Grimm2002). However, this study has identified multiple new strains of P. hermaphrodita, whose pathogenicity, it is recommended, requires examination and testing against A. vulgaris.
Apart from the generalist nematode, A. appendiculatum (Laznik et al., Reference Laznik, Ross, Tóth, Lakatos, Vidrih and Trdan2009), no specialist parasitic nematode of A. vulgaris has yet been recorded. A large number of such slugs that were collected by Ivanova (unpubl.) from the East Flanders province of Belgium were found to be free of infection, contrary to the state of many other local slug species. However, the results of the present study showed that, of all the slug species detected in Norway, nematode diversity, prevalence and intensity was highest in A. vulgaris (table 1). The enemy-release hypothesis suggested an explanation for the success of an invasive species by the lack of its co-evolved natural enemies on the invaded territory (Torchin et al., Reference Torchin, Lafferty and Kuris2001; Ross et al., Reference Ross, Ivanova, Severns and Wilson2010a). Although the parasite burden is high for A. vulgaris in Norway, studies conducted by Ross & Haukeland (unpubl.) have witnessed prevalence levels of 80–90% in the native range.
The role of parasites in the successful invasion of A. vulgaris has not been discussed in the current paper, because nematodes, such as angiostomatids, have little pathogenic impact on their hosts. Obviously, other traits of invasiveness, such as vigour and abundance of A. vulgaris, are to be considered as being of relatively high significance (Hatteland et al., Reference Hatteland, Solhøy, Schander, Skage, Von Proschwitz and Noble2015). However, a tendency to be less affected by enemies is considered as being one of the few consistent characteristics of invasiveness (Colautti et al., Reference Colautti, Grigorovich and MacIsaac2006). Whether A. vulgaris is the exception to the rule remains to be determined.
Acknowledgements
We would like to thank the local farmers, garden centres and private gardeners for their cooperation in the project. We also wish to thank the University of Aberdeen, the Russian Academy of Science, the University of Bergen and Bioforsk for their support. Furthermore, we are grateful to Pascale Metais and Karin Westrum for help with the collecting of slugs.
Financial support
This study was funded by the Research Council of Norway (Yggdrasil, grant number 210948 and project number 803194).
Conflict of interest
None.
