Introduction
Terrestrial gastropod molluscs (slugs and snails) (Mollusca: Gastropoda) are among the most damaging pests in residential gardens, nurseries, greenhouses and agriculture (Godan, Reference Godan1983; DeAngelis, Reference DeAngelis1993; Barker, Reference Barker2002). Current control measures rely on chemical molluscicides, primarily methiocarb, metaldehyde, iron phosphate and sodium ferric EDTA, either formulated into baited pellets or a liquid spray (Hata et al., Reference Hata, Hara and Hu1997; Bailey, Reference Bailey and Barker2002; Henderson & Triebskorn, Reference Henderson, Triebskorn and Barker2002). To date, the aforementioned methods have proven to be the most cost-effective approach for controlling molluscs; however, studies have demonstrated that methiocarb and metaldehyde are toxic to non-target organisms (Purves & Bannon, Reference Purves and Bannon1992; Fletcher et al., Reference Fletcher, Hunter and Barnet1994; Bailey, Reference Bailey and Barker2002). Methiocarb has been banned in many countries but it still can be used in the US as an emergency molluscicidal treatment in ornamental nurseries. There has been increased pressure from regulatory bodies in regions like Europe to ban or limit the use of metaldehyde due to concerns relating to drinking water (Castle et al., Reference Castle, Mills, Gravell, Jones, Townsend, Cameron and Fones2017) and the impact on birds and small mammals (Ross, Reference Ross2019). Metaldehyde was to be banned in Great Britain in spring 2020, but the ban was overturned by the High Court in 2019. Thus, a more effective and environmentally favourable method of mollusc control is needed. Nematodes offer a potential solution as they play an important role in the regulation of gastropod populations in nature (Morand et al., Reference Morand, Wilson, Glen and Barker2004), yet the association is not well studied relative to entomopathogenic nematodes (Wilson & Grewal, Reference Wilson, Grewal, Grewal, Ehlers and Shapiro-Ilan2005).
Most surveys on nematodes associated with terrestrial slugs have, to date, focused on Australia, New Zealand, Europe, Africa and the USA (Ross et al., Reference Ross, Ivanova, Hatteland, Brurberg and Haukeland2016; Wilson et al., Reference Wilson, Wilson, Aalders and Tourna2016). Thus far, nematodes belonging to nine families have been found in association with slugs as phoretic or definitive hosts. These include: Agfidae, Alloionematidae, Angiostomatidae, Alaninematidae, Ascaridae, Cosmocercidae, Diplogastridae, Mermithidae and Rhabditidae (Pieterse et al., Reference Pieterse, Malan and Ross2017). The nematode that has gained greatest interest due to its commercialization as a biological control agent is Phasmarhabditis hermaphrodita (Schneider) Andrássy. Phasmarhabditis hermaphrodita is a facultative parasite capable of infecting slug species from the families Agriolimacidae, Arionidae, Milacidae, Limacidae and Veronicellidae (Wilson et al., Reference Wilson, Glen and George1993; Grewal et al., Reference Grewal, Grewal, Tan and Adams2003; Rae et al., Reference Rae, Verdun, Grewal, Robertson and Wilson2007), as well as various snail species (Rae et al., Reference Rae, Verdun, Grewal, Robertson and Wilson2007; Mc Donnell et al., Reference Mc Donnell, Tandingan De Ley and Paine2018a, Reference Mc Donnell, Lutz, Howe and Denverb). This slug-parasitic nematode is commercially available under the trade names Nemaslug® (BASF) and SlugTech® (Dudutech) in numerous European countries. However, these products are not legally available to date in North America as P. hermaphrodita, as well as other Phasmarhabditids (Phasmarhabditis californica and Phasmarhabditis papillosa) have only recently been reported in California (Tandingan De Ley et al., Reference Tandingan De Ley, McDonnell, Lopez, Paine and De Ley2014; Tandingan De Ley et al., Reference Tandingan De Ley, Holovachov, Mc Donnell, Bert, Paine and De Ley2016) and Oregon (Mc Donnell et al., Reference Mc Donnell, Tandingan De Ley and Paine2018a, Reference Mc Donnell, Lutz, Howe and Denverb), and there is little information on the host range of these US strains.
European pestiferous slugs are a growing problem in Canada, yet there exist few comprehensive studies on the abundance and diversity of slugs in the region (Hawkins et al., Reference Hawkins, Lankester, Lautenschlager and Bell1997; Moss & Hermanutz, Reference Moss and Hermanutz2010; L'Heureux & Angers, Reference L'Heureux and Angers2018). The widespread success of invasive slugs is likely due to a number of factors, including favourable climates and lack of associated natural enemies (Ross et al., Reference Ross, Ivanova, Severns and Wilson2010). In a recent study, P. californica was isolated for the first time from a slug specimen in Edmonton, Alberta (Brophy et al., Reference Brophy, Howe, Denver and Luong2020). This discovery was part of a broader survey conducted in and around Edmonton with the hope of isolating a potential biological control agent of invasive slugs in Canada. Here we present data from the survey of slugs and their associated nematodes collected from residential, agricultural and horticultural sites.
Materials and methods
Slug collection and identification
Slugs were collected from residential gardens, nurseries, greenhouses and agricultural sites in the Edmonton area of Alberta, Canada. The survey was conducted in 2019 between May and October. Slugs were collected by hand and transferred to plastic containers with preformatted lids to allow for air circulation. Once the slugs were returned to the laboratory, they were maintained in an incubator (18°C–12°C, 12 h light:12 h dark cycle) and fed ad lib carrot slices or lettuce until processing. Specimens from the same collection site were kept together to avoid cross-contamination. Slugs were initially identified through morphological examination (Forsyth, Reference Forsyth2004; Mc Donnell et al., Reference Mc Donnell, Paine and Gormally2009; Grimm et al., Reference Grimm, Forsyth, Schueler and Karstad2009; Vlach, Reference Vlach2016; https://idtools.org/id/mollusc/index.php). A molecular diagnosis was only performed on specimens when examination of external morphology and internal genital morphology was inconclusive – for example, immature specimens. A piece of tail tissue was also taken from select slug specimens and placed in 95% ethanol for molecular analysis. DNA was extracted from a small piece (~2 mm × 2 mm) of this tissue using an Omega BioTek blood and tissue kit (Norcross, GA, USA). Partial fragments of the mitochondrial cytochrome c oxidase subunit I (cox 1) gene were sequenced from four of the specimens (table 1) following the methods of Reich et al. (Reference Reich, Gormally, Allcock, Mc Donnell, Castillejo, Iglesias, Quinteiro and Smith2015) using the Folmer et al. (Reference Folmer, Black, Hoeh, Lutz and Vrijenhoek1994) cox 1 primer set LCO-1490 (5′-GGTCAACAAATCATA AAGATATTGG-3′) and HCO-2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′). After trimming and editing with Bioedit v 7.0.5.3 (Hall, Reference Hall1999), the resulting sequences were 648–652 bp long. The closest match to specimens from the Barcode of Life (BOLD) database (Ratnasingham & Hebert, Reference Ratnasingham and Hebert2007) and GenBank (Benson et al., Reference Benson, Cavanaugh, Clark, Karsch-Mizrachi, Lipman, Ostell and Sayers2013) database were then used to confirm our morphological identifications. The sequences from the current study have been deposited in GenBank under accession numbers (table 1).
Table 1. Number and prevalence of slugs associated with nematodes.
Species with a superscript letter indicate that morphological identifications were confirmed using partial COI gene sequences. This was achieved by finding the closest match on the Barcode of Life (BOLD) and GenBank databases.
a Prophysaon andersoni (GenBank accession number: MT680916): 97.82% match with a specimen from British Columbia, Canada, on GenBank and 100% match with a specimen from an unspecified location on BOLD.
b Deroceras laeve, two specimens: (1) (GenBank accession number: MT680915) 100% match with a specimen from British Columbia, Canada on GenBank and 100% match with a specimen from Manitoba, Canada, on BOLD; (2) (GenBank accession number: MT680918) 99.38% match with a specimen from an unspecified location on GenBank and 100% match with a specimen from West Gloucestershire, UK, on BOLD.
c Ambigolimax valentianus (GenBank accession number: MT680917): 99.07% match with a specimen from Aude, France, on GenBank and 99.84% match with a specimen from Antrim, Northern Ireland, on BOLD.
Nematode identification
Slugs were decapitated and cadavers were incubated for two weeks in individual Petri dishes, which were covered and sealed with Parafilm (Wilson et al., Reference Wilson, Wilson, Aalders and Tourna2016). If nematodes were present on the cadaver at that time, three individuals were transferred with a sterile pin to 95% ethanol and later identified using molecular techniques. After removal of the storage ethanol, nematodes were transferred to a proteinase-K-based lysis buffer for DNA extraction as previously described (Williams et al., Reference Williams, Schrank, Huynh, Shownkeen and Waterston1992). The nematode species were identified using 18S ribosomal DNA amplification with the primer set 18A (5′-AAAGATTAAGCCATGCATG-3′) and 26R (5′-CATTCTTGGCAAATGCTTTCG-3′), resulting in an ~800 bp segment, which was directly end sequenced using primer 18A (Blaxter et al., Reference Blaxter, De Ley and Garey1998). All 18S rRNA sequences were compared against GenBank's non-redundant (nr) database using blastn, NCBI Resource Coordinators. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res.2018;46(D1):D8-D13. doi:10.1093/nar/gkx1095. Nematode BLAST matches with percent identity of 98–100 were used for species identification. One representative sequence for each nematode species identified in this study was submitted to GenBank (see table 2 for accession numbers).
Table 2. Nematode species, accession numbers and associated slug species.
ª One representative sequence was submitted to GenBank for each nematode species identified in this study.
Results
A total of 2406 slugs were collected from Edmonton and surrounding areas. Specimens were collected from 72 residential gardens (harvested by homeowners and donated to the lab), four natural green spaces (parks, ravines), four seasonal nurseries and the University of Alberta Botanical Garden (exterior gardens and two year-round greenhouses). Slugs were also opportunistically obtained from two sites adjacent to agricultural fields, a strawberry patch in Thorsby and a ditch alongside a canola field in Falun. The vast majority of slugs were collected from residential gardens, due largely to the overwhelming cooperation of residential gardeners. In total, nine slug species were identified (table 1), representing three slug families (Arionidae, Agriolimacidae and Limacidae). Although we did not conduct a formal population estimate, the peak slug recovery (especially from home gardeners) occurred in August, suggestive of a seasonal peak in the population density of adult slugs (fig. 1).
Fig. 1. Number of slugs collected in the 2019 survey between June and October.
Nematodes were recovered from 20 of the 82 sites surveyed (24.4%). Of all slugs examined, 3.74% were associated with nematodes. The highest prevalence occurred in year-round greenhouses, where 31.0% of the slugs harboured nematodes; only 3.27% of the nearly 2000 slugs from residential gardens harboured nematodes (fig. 2). Deroceras reticulatum was the most common slug, but these were rarely associated with nematodes (3.20%; table 1). By contrast, nematodes were recovered from 16 of 65 (24.6%) specimens of Ambigolimax valentianus, most of which were collected from the year-round greenhouses (table 1). A total of seven nematode species were identified, plus four specimens that could only be identified to genus level (table 2). The two most common nematodes were Caenorhabditis elegans and Pelodera pseudoteres. The facultative parasite P. californica was recovered from a single Arion rufus specimen, collected from a potted plant located on the exterior grounds of a seasonal nursery (Brophy et al., Reference Brophy, Howe, Denver and Luong2020).
Fig. 2. Prevalence of nematodes in all slugs surveyed based on the type of collection site. Numbers represent sample sizes.
Discussion
This study is the first survey of nematodes associated with slugs in Canada, and specifically in the central Alberta region. A total of nine slug species and 11 nematode taxa (seven to species level; four to genus level) were identified. Overall, the nematodes represented four families: Alloionematidae, Diplogastridae, Panagrolaimidae and Rhabditidae. The most common family Rhabditidae is a large family with many genera associated with slugs, including Rhabditis, Caenorhabditis and Phasmarhabditis (Grewal et al., Reference Grewal, Grewal, Tan and Adams2003; Pieterse et al., Reference Pieterse, Malan and Ross2017). Most of the nematodes we recovered were likely either free-living or phoretic on the slugs, with the exception of P. californica, which is a facultative parasite of slugs (Tandingan De Ley et al., Reference Tandingan De Ley, Holovachov, Mc Donnell, Bert, Paine and De Ley2016). The reason for this may be due to the use of the decapitation technique (Wilson et al., Reference Wilson, Wilson, Aalders and Tourna2016), which can encourage the replication of free-living or phoretic nematodes. In the future, slug cadavers should be rinsed thoroughly prior to placing in Petri dishes to remove phoretic species. The two most common nematodes, C. elegans and P. pseudoteres, are considered free-living or phoretic on arthropods, slugs and earthworms (Schulte, Reference Schulte1989; Kiontke & Sudhaus, Reference Kiontke and Sudhaus2006). However, C. elegans has been shown to persist in the intestine of molluscs (Petersen et al., Reference Petersen, Hermann, Barg, Schalkowski, Dirksen, Barbosa and Schulenburg2015). Mollusc-associated nematodes have evolved independently four times (in the Nematoda) in two groups: Metastrongyloidea (Order Strongylida), most of which use molluscs as intermediate hosts, and the Rhabditoidea (Order Rhabditida), which primarily use molluscs as definitive hosts (Grewal et al., Reference Grewal, Grewal, Tan and Adams2003).
The invasive slug D. reticulatum was the most common slug in our survey, and is the most widespread and important pest in Canada (Grimm et al., Reference Grimm, Forsyth, Schueler and Karstad2009). In spite of the over-representation of this species in our survey, the majority of which were collected from residential gardens, only 3.2% were found in association with nematodes. Previous studies have recovered nematodes from 3 to 6% of all slugs surveyed in the USA (Ross et al., Reference Ross, Ivanova, Severns and Wilson2010), South Africa (Ross et al., Reference Ross, Ivanova, Sirgel, Malan and Wilson2012) and Norway (Ross et al., Reference Ross, Ivanova, Hatteland, Brurberg and Haukeland2016). Nearly a quarter (24.6%) of the Ambigolimax valentianus slugs we collected harboured nematodes, most of which were collected from year-round greenhouses. Similarly, a study in Norway found that whilst the prevalence of infection was 18.7% among all slugs, 34.8% of Arion vulgaris examined were associated with nematodes (Ross et al., Reference Ross, Ivanova, Hatteland, Brurberg and Haukeland2016).
All slug species, except Prophysaon andersoni (native), in our survey are introduced and of European origin. It is also important to note that the Holarctic Deroceras laeve is thought to have both introduced and native populations in the US (Mc Donnell et al., Reference Mc Donnell, Paine and Gormally2009) and a similar population structure likely exist in Canada. The economic and ecological impact of introduced molluscs on native species and habitats is not well understood in Canada. Introduced temperate species, such as D. reticulatum and Arion spp., are considered fairly intolerant of prolonged freezing temperatures (Storey et al., Reference Storey, Storey and Churchill2007). Yet, D. laeve have been reported from Baffin Island in north-eastern Canada, known for bitterly cold winters and short summers (Storey et al., Reference Storey, Storey and Churchill2007; Grimm et al., Reference Grimm, Forsyth, Schueler and Karstad2009). Further research is needed to better understand how these temperate species survive winter in Alberta, particularly outside greenhouses.
Our discovery of the nematode P. californica in Canada is especially exciting because it is a facultative parasite and, therefore, should be tested for its potential to be a biological control agent. The congeneric P. hermaphrodita is commercially available in Europe as a bio-molluscicide; however, to date, this nematode cannot be sold in Canada as it has only recently been identified in the region (Brophy et al., Reference Brophy, Howe, Denver and Luong2020). Notably, P. californica was first described in association with a slug in California (Tandingan De Ley et al., Reference Tandingan De Ley, Holovachov, Mc Donnell, Bert, Paine and De Ley2016) and has recently been collected in New Zealand (Wilson et al., Reference Wilson, Wilson, Aalders and Tourna2016) and Ireland (Carnaghi et al., Reference Carnaghi, Rae and Tandingan De Ley2017). Future studies are, therefore, needed to understand the virulence and biocontrol potential of P. californica, as well as the risk to non-target species.
In our study, we sampled opportunistically from a variety of sites, and, as such, did not achieve equal sampling from the various habitat types. Future surveys should increase sampling of molluscs from nurseries and greenhouses where high prevalence of nematodes were observed. In addition, horticultural sites, natural habitats and agricultural crops should be surveyed more intensively in future studies since the extent of the economic and ecological impact of invasive slugs is poorly understood. Moreover, a systematic survey of the abundance and diversity of nematodes associated with slugs in Canada is greatly needed. Lastly, if P. californica or other species of Phasmarhabditids are to be pursued as biological control agents in Canada, it is critical that their host range be determined, particularly their potential impacts on native gastropods.
Acknowledgements
Our thanks to all the residential gardeners, local growers, garden centres and nurseries that participated in the survey. We are also grateful to the undergraduate volunteers, K. Kent, L. MacLeod and G. Mulyk, for assisting with the collection and monitoring of slugs.
Financial support
This research was funded by Alberta Environment and Parks. J.L.R. was supported by the Nuffield Farming Scholarship Trust.
Conflicts of interest
None.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. 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.
