Angiostoma norvegicum n. sp. (Nematoda: Angiostomatidae) a parasite of arionid slugs in Norway

Angiostoma norvegicum n. sp. (Angiostomatidae) is described from the oesophagus, crop and the buccal mass of five species of slugs of the family Arionidae, Arion vulgaris (Moquin-Tandon), Arion ater (L.), Arion fasciatus (Nilsson), Arion fuscus (Müller) and Arion rufus/Arion ater hybrid), collected throughout Norway. Angiostoma norvegicum n. sp. was found parasitising arionids at seven of the 30 sample sites examined (23.3%), and 9.9% of all Arion spp. were infected with this nematode. The new species is characterised by its large size (4.0–8.6 mm long) and in having: lateral alae; 6 + 6 papillae at the cephalic end; a large circular mouth aperture; a spacious stoma; a pharyngeal basal bulb without valvular apparatus; an excretory pore near the base of bulb; a distal part of posterior ovary always outstretched; an anterior ovary distally nearly always outstretched; a vulva situated anterior to mid-body; long, nearly straight spicules and a small gubernaculum; three circumcloacal papillae and caudal genital papillae (GP) arranged in a pattern 1+2/3+3 with GP 5 and GP 8 opened on dorsal side of narrow bursa not reaching tail tip; short conical tails in both sexes with tips supplied by 4 short, unequal denticles. Morphologically, A. norvegicum n. sp. is similar to Angiostoma limacis Dujardin, 1845, which diagnostic characteristics are given based on examination of specimens from Norway and the UK. Conversely, the phylogenetic analyses based on D2D3 large subunit (LSU) rRNA gene sequences performed in the present study did not support the morphological affinity of these two species. Phylogenetic analyses demonstrated that although Angiostoma spp. cluster together, A. norvegicum n. sp. forms a tight monophyletic clade with the milacid nematode parasites Angiostoma margaretae Ross, Malan & Ivanova, 2011 and Angiostoma milacis Ivanova & Wilson, 2009.

bulb; a distal part of posterior ovary always outstretched; an anterior ovary distally nearly always outstretched; a vulva situated anterior to mid-body; long, nearly straight spicules and a small gubernaculum; three circumcloacal papillae and caudal genital papillae (GP) arranged in a pattern 1?2/3?3 with GP 5 and GP 8 opened on dorsal side of narrow bursa not reaching tail tip; short conical tails in both sexes with tips supplied by 4 short, unequal denticles. Morphologically, A. norvegicum n. sp. is similar to Angiostoma limacis Dujardin, 1845, which diagnostic characteristics are given based on examination of specimens from Norway and the UK. Conversely, the phylogenetic analyses based on D2D3 large subunit (LSU) rRNA gene sequences performed in the present

Materials and methods
Slug collection Arion spp. were collected from 30 sample sites in Norway. Habitats included roadside verges, domestic gardens and agricultural land. Localities were found in cooperation with local advisory services and coordinates were recorded. Slugs were collected during late August, September and October 2011, and identified through morphological examination, dissection of genitalia and analysis of mitochondrial DNA . Specimens of A. limacis used for illustrations of details of diagnostic value were collected from Arion distinctus Mabille in Scotland by E. Ivanova in 2006Ivanova in -2007.

Morphological analysis
Nematodes were washed with Ringer's solution from the dissected host into a watch glass and then picked with a needle. Depending on the intensity of infection, between one and four nematodes collected from each species of Arion was stored in 70% ethanol for DNA extraction. The remainder of nematodes were fixed by adding hot 4-5% formaldehyde for morphological studies. Measurements and drawings were taken from formaldehyde-fixed nematodes mounted on permanent slides after processing to glycerine following Seinhorst (1959). All measurements are in micrometres, and presented in the text as the range followed by the mean in parentheses. The following abbreviations were used: L, body length; V%, distance from anterior extremity to vulva as a percentage of body length a, b, c, indices of De Man. Genital papillae formula is given as proposed by Sudhaus & Fürst von Lieven (2003). For light microscopy, compound microscopes Zeiss Jenaval and Nikon Eclipse E200 with drawing attachment were used. Illustrations were finalised with WACOM Intuos A4 USB drawing tablet and Adobe Illustrator CS5 following Coleman (2003). For scanning electron microscopy (SEM), nematodes were rehydrated after formaldehyde, dehydrated in a graded ethanol series, critical-point dried using a HCP-2 HITACHI dryer, mounted on aluminium stubs and coated with gold in a BIO-RAD SC502 sputter coater. Specimens were studied in a JCM-6380 LA SEM and CamScan S2 (Cambridge Instruments, UK).

Results
General data on the occurrence of A. norvegicum n. sp. and other slug-parasitic nematodes in Norway were given in Ross et al. (2016) where the new species was designated as Angiostoma sp. A total of 322 Arion spp. were collected from 30 sample sites around Norway. Angiostoma norvegicum n. sp. was found parasitising five out of seven species of Arionidae at 23.3% of sample sites (Table 1). Of all Arion spp. collected, 9.9% were infected with A. norvegicum n. sp., which was isolated from the oesophagus, crop and buccal mass of slug hosts. The number of A. norvegicum n. sp. recovered from Arion spp. varied from 1 to 56 nematodes, with a sex ratio of approximately 2 females: 1 male.

Remarks
The new species is characterised by its large size and the presence of: lateral alae; a large circular mouth aperture; a spacious stoma; a club-shaped pharynx with a basal bulb without valvular apparatus; a distal part of posterior ovary always outstretched; an anterior ovary distally nearly always outstretched; long, nearly straight spicules and a small gubernaculum; three circumcloacal papillae; caudal genital papillae arranged in a pattern 1?2/3?3 with GP 5 and GP 8 opened on dorsal side of narrow bursa not reaching tail tip; short conical tails in both sexes with tips supplied by 4 short, unequal denticles, and parasitising Arion spp. (Table 1). This new species is morphologically very similar to A. limacis, another parasite of the Arionidae, in the body proportions, the shape and structure of the stoma, the shape of the tail in both sexes and the shape and size of spicules and gubernaculum (Mengert, 1953;Morand & Spiridonov, 1989) (Fig. 5C-D). It can be distinguished from the latter by having a shorter stoma (mean 14-17 vs 30 lm), the presence (vs absence) of lateral alae, the presence (vs absence) of a prominent precloacal flap in males, distally outstretched ovaries (always posterior and in majority of cases, also anterior ones) (vs reflexed ovaries). Slight differences between these species include thinner stoma walls (mean 3 lm in A. norvegicum n. sp. vs 5 lm in A. limacis) and different proportions of stoma (in A. norvegicum n. sp., its bowl-shaped portion is shorter while a funnel-shaped one is longer than in A. limacis) and pharynx (in A. norvegicum n. sp., a procorpus is typically slightly expanded at ends while in A. limacis, it is usually uniformly wide with occasional slightest expansion at posterior; also, an isthmus is remarkably much shorter in A. limacis, along with the smaller bulb) (Fig. 4A). Also, the head end in the new species is without the slight constriction at the level of stoma base present in A. limacis (Fig. 5A-B).
The tail shape and the presence of lateral alae in the new species represent similarities to A. aspersae Morand, 1986. However, A. norvegicum differs by lacking a valvular apparatus in the basal bulb and paired papilliform appendages in front of cloaca, and by having denticles on the tail tip, 9 pairs of genital papillae (vs 10), longer spicules (mean 119 vs 80 lm) and a much larger stoma (Morand, 1986).

Molecular differentiation and phylogenetic relationships
Identical tree topologies were obtained from maximum likelihood (ML), maximum parsimony (MP) and distance analyses. Therefore only the maximumlikelihood tree is presented (Fig. 6) along with bootstrap support from each method of analysis. Phylogenetic analyses demonstrated that the majority of Angiostoma spp. cluster together, although only under weak bootstrap support (52/51/53), leaving A. glandicola as a sister group to the other Angiostoma spp. Within this group, A. norvegicum n. sp. formed a monophyletic clade with the two milacid parasites, A. margaretae and A. milacis, under strong bootstrap support (96/96/95).

Discussion
The new species, A. norvegicum n. sp., is described from five Arion slug hosts (Arion ater, A. fasciatus, A. fuscus, A. vulgaris and Arion rufus/Arion ater hybrid) collected throughout Norway. The nematode was isolated from the oesophagus, crop and the buccal mass of its hosts, thus differing somewhat from other Angiostoma spp. that have been isolated from the intestine (Mengert, 1953;Spiridonov, 1985;Morand, 1988Morand, , 1992Korol & Spiridonov, 1991;Morand & Barker, 1995), hepatopancreas , oesophagus (Ivanova & Wilson, 2009;Ross et al., 2011) and pallial cavity (Morand, 1986) of gastropod hosts.   Angiostoma norvegicum n. sp. is extremely prevalent with results similar to that of another arionidassociated species, A. limacis from the UK (Ivanova & Wilson, 2009), however mean intensity of infection with A. norvegicum n. sp. is higher in all infected host species and particularly in the type-host, A. vulgaris. This species also known as the Iberian or Spanish slug, is highly invasive and its current distribution includes mainland Europe, UK, Ireland and USA (Weidema, 2006). However in its native range, which is assumed to be the Iberian Peninsula and the South-West of France, it is much less numerous than in invaded territories (von Proschwitz & Winge, 1994). The species is also known under the name of a 'killer slug' because of its habit of feeding on dying and dead slugs. Arion vulgaris has the ability to hybridise with the related species of Arion thus posing a threat to the native biota (Hatteland et al., 2015). In Norway, the slug was first recorded in 1988 (Hatteland et al., 2013), and is known to inflict serious damage in gardens, horticulture and agriculture.
The limited number of natural enemies of the notorious pest A. vulgaris was always considered as one of the traits supporting its success in colonisation of new territories. This study has shown that in Norway A. vulgaris does not lack natural enemies (at least regarding nematodes) and even shows a greater susceptibility to slug-associated nematodes than native slug species. This fact can point on the limited ability of the host to tolerate nematode invasion in the conditions of the certain area. The presence of the heavy, combined nematode infection (A. appendiculatum, A. flexilis, A. limacis, A. norvegicum n. sp. and P. hermaphrodita) of the invasive slug species on the northern boundary of its distribution, together with its absence closer to its native range , is Maximum-likelihood (ML) phylogenetic tree based on D2D3 LSU rRNA gene sequence data for several Angiostoma spp. along with selected species of the Rhabditidae used as the outgroup. Phylogenetic analysis of 496 unambiguously aligned nucleotide positions used the GTR correction model with eight gamma-rates and invariable sites. Bootstrap support was calculated based on 1,000 replicates using maximum-likelihood, distance and maximum parsimony methods respectively. Only bootstrap values above 65% are shown in opposition to the hypothesis of parasite release, which attributes the success in the establishment of an invader on invaded areas to its release of the natural enemies.
The prevalence of A. norvegicum n. sp. in A. vulgaris was at the same level as of other larger arionids, i.e. A. ater and A. ater/A. rufus hybrids. The maximum intensity of nematodes per host (56) was the highest ever recorded for a species of the Angiostomatidae. However the absence of A. norvegicum n. sp. at certain study sites indicates that the infection of A. norvegicum n. sp. was first based on native larger arionids, i.e. A. ater and A. ater/A. rufus. It can be assumed that A. norvegicum n. sp. was transmitted to A. vulgaris through feeding on other infected Arion spp.
Members of the genus Angiostoma are not strictly host-specific, however the infection of certain nematode species tends to be based on related host species (Ivanova et al., 2013). In regard to Arionidae, A. limacis parasitises arionids and, rarely, agriolimacids of the genus Deroceras Rafinesque (see Morand, 1988;Ivanova & Wilson, 2009). In the description of A. dentiferum, Mengert (1953) indicated two host species, Limax cinereoniger Wolf and Arion subfuscus (Draparnaud), from the families Limacidae and Arionidae, respectively. However further studies demonstrated the presence of A. dentiferum in Limax spp. slugs only (Morand, 1988;Ivanova et al., 2013). Morphologically, A. dentiferum is remarkably different from both species of Angiostoma parasitising arionid slugs. Thus, A. norvegicum n. sp. is the second member of Angiostoma associated with slugs of the family Arionidae. Both species, A. limacis and A. norvegicum n. sp., are very close morphologically. Conversely, the phylogenetic analyses performed in the present study did not support the morphological affinity of these two species. Phylogenetic analyses showed that the majority of Angiostoma spp. clusters together, although only under weak bootstrap support, leaving A. glandicola as a sister group to the other Angiostoma spp. Within this group, A. norvegicum n. sp. formed a monophyletic clade with the two milacid parasites, A. margaretae and A. milacis, thus supporting certain morphological similarities (stoma shape, and the presence of lateral alae, distally outstretched ovaries).
The identification of A. norvegicum n. sp. brings the total complement of the genus to 18 species, 14 from terrestrial molluscs and four from amphibian and reptile hosts (Falcón-Ordaz et al., 2008). It has been argued that members of the genus Angiostoma are parasites of vertebrates and use molluscs as obligate intermediate hosts (Grewal et al., 2003). However, to date, no Angiostoma spp. have been recorded from both invertebrate (supposedly intermediate) and vertebrate (definitive) hosts. In addition, Angiostoma spp. parasitic in both molluscan and vertebrate hosts have been isolated in their adult stages, indicating that they are in their final phase of development. The resolution of the genus can only be confirmed through molecular and phylogenetic analysis of the genus; however, to date insufficient molecular data are available for Angiostoma spp. from vertebrate hosts.