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Nematodes and trematodes associated with terrestrial gastropods in Nottingham, England

Published online by Cambridge University Press:  02 November 2022

P. S. Andrus Open the ORCID record for P. S. Andrus [Opens in a new window] ,
R. Rae Open the ORCID record for R. Rae [Opens in a new window]  and
C. M. Wade
P. S. Andrus
Affiliation:
School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UK
R. Rae
Affiliation:
School of Biological and Environmental Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK
C. M. Wade*
Affiliation:
School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UK
*
Author for correspondence: C. M. Wade, E-mail: [email protected]
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Abstract

A parasitological survey of terrestrial slugs and snails was conducted at popular dog walking locations across the city of Nottingham, with the intensions of finding gastropods infected with parasites of medical (or veterinary) importance such as lungworm (metastrongyloid nematodes) and trematodes. A total of 800 gastropods were collected from 16 sites over a 225 km2 area. The extracted nematodes and trematodes were identified by molecular barcoding. Of the 800 gastropods collected, 227 were infected (172 had nematode infections, 37 had trematode infections and 18 had both nematode and trematode infections). Of the nematode infected gastropods genotyped, seven species were identified, Agfa flexilis, Angiostoma gandavense, Angiostoma margaretae, Cosmocerca longicauda, Phasmarhabditis hermaphrodita, Phasmarhabditis neopapillosa and an unknown Cosmocercidae species. Of the trematode infected gastropods genotyped, four species were identified, Brachylaima arcuate, Brachylaima fuscata, Brachylaima mesostoma and an unknown Plagiorchioidea species. No lungworm species were found within the city of Nottingham. To our knowledge, this study represents the first survey of gastropod-associated nematodes and trematodes in the East midlands of the United Kingdom.

Keywords

lungwormsnematodestrematodesparasitologygastropods
Type
Research Paper
Information
Journal of Helminthology , Volume 96 , 2022 , e81
Creative Commons
Creative Common License - CCCreative Common License - BY
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited.
Copyright
Copyright © The Author(s), 2022. Published by Cambridge University Press

Introduction

Slugs and snails (Class: Gastropoda) comprise approximately 35,000 extant species and can host a diverse range of metazoan parasites (and parasitoids) such as cestodes, trematodes, nematodes, insects and acarids (Barker & Efford, Reference Barker, Efford and Barker2004; Chapman, Reference Chapman2009). There are approximately 25,000 extant species of nematodes, of which 3500 are parasites of invertebrates (Grewal et al., Reference Grewal, Grewal, Tan and Adams2003). Of these, 50 metastrongyloid (lungworms) species are of medical or veterinary importance, with notable genera being Aelurostrongylus, Angiostrongylus, Crenosoma, Elaphostrongylus, Muellerius, Neostrongylus, Oslerus, Prostrongylus and Troglostrongylus (Alicata, Reference Alicata1965; Skorping et al., 1980; Campbell et al., 1988; Diez-Baños et al., 1989; Schjetlein et al., 1995; Majoros et al., Reference Majoros, Fukár and Farkas2010; Panayotova-Pencheva, 2011; Kim et al., Reference Kim, Hayes, Yeung and Cowie2014; Patel et al., Reference Patel, Gill, Fox, Hermosilla, Backeljau, Breugelmans, Keevash, McEwan, Aghazadeh and Elson-Riggins2014; Conboy, 2015; Helm et al., Reference Helm, Roberts, Jefferies, Shaw and Morgan2015; Aziz et al., Reference Aziz, Daly, Allen, Rowson, Greig, Forman and Morgan2016; Hadi, 2018; Hicklenton & Betson, Reference Hicklenton and Betson2019; Penagos-Tabares et al., Reference Penagos-Tabares, Groß, Hirzmann, Hoos, Lange, Taubert and Hermosilla2020). Nematodes have evolved diverse relationships with gastropods, with some species using them as an intermediate host (e.g. juveniles of lungworm species) while others (Rhabditidae, Mermithidae and Ascarididae) parasitize gastropods and use them as their definitive host; or for other means such as necromeny or transportation (paratenic) (Grewal et al., Reference Grewal, Grewal, Tan and Adams2003; Ivanova et al., Reference Ivanova, Clausi, Sparacio and Spiridonov2019).

Digenetic trematodes comprise approximately 40,000 extant species, with more than 18,000 described species (Cribb et al., Reference Cribb, Bray, Littlewood, Pichelin and Herniou2001; Kostadinova & Pérez-del-Olmo, Reference Kostadinova and Pérez-del-Olmo2014). Unlike nematodes, digenetic trematodes use invertebrates exclusively as an intermediate host, with a vertebrate (typically a fish, mammal, or bird) being used as their definitive host (Barker & Efford, Reference Barker, Efford and Barker2004). Notable genera of medical or veterinary importance are Clonorchis, Fasciola, Fasciolopsis, Gastrodiscoides, Heterophyes, Metagonimus, Opisthorchis, Paragonimus and Schistosoma (Doughty, Reference Doughty1996; Kostadinova & Pérez-del-Olmo, Reference Kostadinova and Pérez-del-Olmo2014). Trematode species which infect terrestrial gastropods use them in order to infect bird, mammal, or reptile definitive hosts which prey on gastropods (Morley & Lewis, Reference Morley and Lewis2008). Most species specialize in infecting one type of definitive host, but some species can infect multiple hosts (Butcher & Grove, Reference Butcher and Grove2005). The lifecycle of these trematodes first involves a gastropod host being infected through the ingestion of eggs (excreted by an infected definitive host). After ingestion, it takes one to three months for asexual sporocysts to produce cercariae within the first intermediate gastropod host (Butcher & Grove, Reference Butcher and Grove2003). Gastropods can act as both the first and second intermediate host, as infected snails (first intermediate) shed cercariae in their mucus which can infect other gastropods through bodily contact (or themselves making them a first and second intermediate host simultaneously) (Butcher & Grove, Reference Butcher and Grove2005). The successful cercariae develop into mature metacercariae after four months and can survive up to another four months within the gastropod host. The transmission cycle is completed when the secondary intermediate gastropod host is ingested by a bird, mammal, or reptile definitive host (Morley & Lewis, Reference Morley and Lewis2008).

The current understanding of nematodes and trematodes associated with terrestrial gastropods in Europe is based on parasitological surveys conducted in Austria (Penagos-Tabares et al., Reference Penagos-Tabares, Groß, Hirzmann, Hoos, Lange, Taubert and Hermosilla2020), Belgium (Singh et al., Reference Singh, Couvreur, Decraemer and Bert2020), Bulgaria (and Crimea) (Ivanova et al., Reference Ivanova, Spiridonov and Panayotova-Pencheva2013), the Czech Republic (Heneberg et al., Reference Heneberg, Sitko and Bizos2016), Denmark (Taubert et al., Reference Taubert, Pantchev, Vrhovec, Bauer and Hermosilla2009), England (Morley & Lewis, Reference Morley and Lewis2008; Patel et al., Reference Patel, Gill, Fox, Hermosilla, Backeljau, Breugelmans, Keevash, McEwan, Aghazadeh and Elson-Riggins2014; Hicklenton & Betson, Reference Hicklenton and Betson2019), France (Nguyen et al., Reference Nguyen, Rossi, Argy, Baker, Nickel, Marti, Zarrouk, Houzé, Fantin and Lefort2017), Germany (Ross et al., Reference Ross, Ivanova, Hatteland, Brurberg and Haukeland2016; Lange et al., Reference Lange, Penagos-Tabares, Hirzmann, Failing, Schaper, Van Bourgonie, Backeljau, Hermosilla and Taubert2018; Gérard et al., Reference Gérard, Ansart, Decanter, Martin and Dahirel2020), Hungary (Majoros et al., Reference Majoros, Fukár and Farkas2010), the Netherlands and Norway (Antzée-Hyllseth et al., Reference Antzée-Hyllseth, Trandem, Torp and Haukeland2020), Poland (Filipiak et al., Reference Filipiak, Haukeland, Zając, Lachowska-Cierlik and Hatteland2020), Italy (Ivanova et al., Reference Ivanova, Clausi, Sparacio and Spiridonov2019), Slovenia (Laznik et al., Reference Laznik, Ross and Trdan2010), Scotland (Helm et al., Reference Helm, Roberts, Jefferies, Shaw and Morgan2015), Spain (Foronda et al., Reference Foronda, López-González and Miquel2010; Jefferies et al., Reference Jefferies, Vrhovec, Wallner and Catalan2010; Paredes-Esquivel et al., Reference Paredes-Esquivel, Sola, Delgado-Serra, Riera, Negre, Miranda and Jurado-Rivera2019; Martín-Carrillo et al., Reference Martín-Carrillo, Feliu and Abreu-Acosta2021) and Wales (Ross et al., Reference Ross, Ivanova, Severns and Wilson2010a, Reference Ross, Ivanova, Spiridonov, Waeyenberge, Moens, Nicol and Wilsonb; Aziz et al., Reference Aziz, Daly, Allen, Rowson, Greig, Forman and Morgan2016). The majority of these studies found no medically important nematode or trematode species, with primarily free-living, gastropod-specific and veterinary important species being reported. Four common lungworm genera (Metastrongyloidea) of medical/veterinary importance were present in Europe (Angiostrongylus, Crenosoma, Aelurostrongylus and Troglostrongylus) with Angiostrongylus (An.) cantonensis the only medically important species reported. Angiostrongylus (An.) cantonensis is a parasite endemic to Asia, the Caribbean and Pacific Islands. In Europe it has been found infecting black rats (Rattus rattus) in the Canary and Balearic Islands and the Algerian hedgehog (Atelerix algirus) in mainland Spain (Foronda et al., Reference Foronda, López-González and Miquel2010; Paredes-Esquivel et al., Reference Paredes-Esquivel, Sola, Delgado-Serra, Riera, Negre, Miranda and Jurado-Rivera2019; Martín-Carrillo et al., Reference Martín-Carrillo, Feliu and Abreu-Acosta2021). Furthermore, Nguyen et al. (Reference Nguyen, Rossi, Argy, Baker, Nickel, Marti, Zarrouk, Houzé, Fantin and Lefort2017) reported the first autochthonous human case of An. cantonensis infection in France. In addition to the metastrongyloids, seven additional gastropod-related nematode families were reported in Europe, the Agfidae, Alloionematidae, Angiostomatidae, Cosmocercidae, Diplogasteridae, Mermithidae and Rhabditidae. The most common genera of trematodes found were Brachylaima, Eurytrema, Michajlovia, Urogonimus and Urotocus. Certain species of Brachylaima (Brachylaimiasis) and Eurytrema (Eurytrematosis) have been found to cause infection within humans in Australia and Brazil, respectively (Schwertz et al., Reference Schwertz, Lucca, da Silva, Baska, Bonetto, Gabriel, Centofanti and Mendes2015; Gracenea & Gállego, Reference Gracenea and Gállego2017) though there have as yet been no reports of human infection in Europe. Trematodes associated with terrestrial gastropods in Europe have not been as well studied as nematodes, most probably due to the majority of these species of medical or veterinary importance being associated with aquatic snail species.

Lungworm nematode infections have been extensively studied in Europe (Taubert et al., Reference Taubert, Pantchev, Vrhovec, Bauer and Hermosilla2009; Patel et al., Reference Patel, Gill, Fox, Hermosilla, Backeljau, Breugelmans, Keevash, McEwan, Aghazadeh and Elson-Riggins2014; Helm et al., Reference Helm, Roberts, Jefferies, Shaw and Morgan2015; Taylor, Reference Taylor2015; Aziz et al., Reference Aziz, Daly, Allen, Rowson, Greig, Forman and Morgan2016; Helm & Morgan, Reference Helm and Morgan2017; Lange et al., Reference Lange, Penagos-Tabares, Hirzmann, Failing, Schaper, Van Bourgonie, Backeljau, Hermosilla and Taubert2018; Elsheikha et al., Reference Elsheikha, Wright, Wang and Schaper2019; Hicklenton & Betson, Reference Hicklenton and Betson2019; Fuehrer et al., Reference Fuehrer, Morelli and Bleicher2020; Penagos-Tabares et al., Reference Penagos-Tabares, Groß, Hirzmann, Hoos, Lange, Taubert and Hermosilla2020). Lungworm infections are fatal to companion animals due to the severe respiratory disease and bleeding disorders caused by the parasite (Taubert et al., Reference Taubert, Pantchev, Vrhovec, Bauer and Hermosilla2009). Angiostrongylus (An.) vasorum and Crenosoma vulpis are widespread across the United Kingdom, with domesticated dogs and red foxes (Vulpes vulpes) acting as their definitive hosts (Helm & Morgan, Reference Helm and Morgan2017). Geography is one of the main risk factors for An. vasorum infections in dogs, with the most endemic areas of the United Kingdom being Southern England and Southern Wales (Patel et al., Reference Patel, Gill, Fox, Hermosilla, Backeljau, Breugelmans, Keevash, McEwan, Aghazadeh and Elson-Riggins2014; Helm & Morgan, Reference Helm and Morgan2017; Hicklenton & Betson, Reference Hicklenton and Betson2019) though An. vasorum in the United Kingdom is spreading northwards, with the parasite already established in Northern England and Scotland (Helm et al., Reference Helm, Roberts, Jefferies, Shaw and Morgan2015; Aziz et al., Reference Aziz, Daly, Allen, Rowson, Greig, Forman and Morgan2016). Reasons for the spread of An. vasorum include a warmer climate which favours the parasites’ development and the urbanization of wild red fox populations acting as a reservoir of infection, with an estimated one in five infected (Taylor, Reference Taylor2015; Helm & Morgan, Reference Helm and Morgan2017). Crenosoma vulpis transmission is similar to An. vasorum but is more commonly reported in wild canid species than domesticated dogs (Lange et al., Reference Lange, Penagos-Tabares, Hirzmann, Failing, Schaper, Van Bourgonie, Backeljau, Hermosilla and Taubert2018). Similarly, Aelurostrongylus (Ae.) abstrusus is a globally distributed lungworm species that infects wild and domesticated cat species, with a prevalence of 1.7% in United Kingdom house cats (Helm & Morgan, Reference Helm and Morgan2017; Elsheikha et al., Reference Elsheikha, Wright, Wang and Schaper2019). Lungworm infections in domesticated cats and dogs are thought to be underreported as some infections can be asymptomatic and milder cases are commonly misdiagnosed as other disorders such as hypersensitivity (Wright, Reference Wright2009; Penagos-Tabares et al., Reference Penagos-Tabares, Lange, Chaparro-Gutiérrez, Taubert and Hermosilla2018; Pohly et al., Reference Pohly, Nijveldt, Stone, Walden, Ossiboff and Conrado2022).

The primary aim of this study was to investigate which species of terrestrial gastropods are commonly found at dog walking sites in the city of Nottingham and the county of Nottinghamshire, to determine which nematode and trematode species are associated with these gastropods and to determine infection rates. The secondary aim was to investigate whether lungworm nematode species that cause veterinary disease are found at popular dog walking sites across the city of Nottingham and the county of Nottinghamshire.

Materials and methods

Collection sites and gastropod identification

Slugs and snails were collected from 16 sites across Nottinghamshire from June to November 2020 and June to November 2021. All sites were popular dog walking locations and included recreational grounds, country parks, public gardens and nature reserves (table 1; fig. 1). A total of 800 gastropods were collected by hand with 50 specimens collected from each site and with a maximum of ten individuals per species being taken. Specimens were identified morphologically using a Terrestrial Mollusc Key (https://idtools.org/id/mollusc/key.php) (White-McLean, Reference White-McLean2011) and the Slugs of Britain and Ireland as an illustrated guide (Rowson et al., Reference Rowson, Turner, Anderson and Symondson2014).

Table 1. Collection sites surveyed across the city of Nottingham and surrounding areas.

Fig. 1. Map of collection sites (n = 16) across the city of Nottingham and surrounding areas (Google 2022).

Gastropod dissection

Specimens were dissected into four equal pieces within 24-h of collection and placed into a 50 ml falcon tube containing Ash's digestion solution (0.7% pepsin in 0.5% hydrochloric acid) for four to eight hours (Ash, Reference Ash1970). The solution was then placed into a 9 cm Petri dish and examined under a dissection microscope for the presence of nematodes, or the metacercariae stage of trematodes. Nematodes were categorized as either juvenile or adult worms. When found, nematodes and metacercariae were individually picked and placed into 0.2 ml tubes containing 70% ethanol (adult worms were separated from juveniles) and stored at −20°C.

DNA extraction, polymerase chain reaction (PCR) amplification and sequencing

The DNA extractions were done on single nematodes or trematodes using a modified CTAB extraction method (Goodacre & Wade, Reference Goodacre and Wade2001). Extracted samples were resuspended in 100 μl of 10 mM (TRIS-HCl, pH 8.0) buffer. A list of extracted and genotyped samples for each site can be found in online supplementary tables 1 and 2. Promega GoTaq® G2 Master Mix buffer was used for all PCR reactions: 1 μl of DNA template was added to 24 μl of 1× Master Mix buffer (1U TAQ, 0.2 μM primers, 200 μM each dNTP, 1.5 mM MgCl2). The nematode DNA samples were identified using the region of the ribosomal RNA spanning the 18S-ITS1-5.8S-ITS2, which was amplified using the universal nematode primer set developed by Nadler et al. (Reference Nadler, Hoberg, Hudspeth and Rickard2000) (N93: 5′-TTG AAC CGG GTA AAA GTC G-3′ and N94: 5′-TTA GTT TCT TTT CCT CCG CT-3′). The trematode DNA samples were identified using the 18S rRNA gene, which was amplified using the universal trematode primer set developed by Kim et al. (Reference Kim, Hong, Ryu, Park, Chae, Choi, Sim and Park2019) (LPF: 5′-AGG GAA TGG GTG GAT TTA TT-3′ and LPR: 5′-AGA CAC GAC TGA AAG GTT GC-3′). The PCR conditions used were an initial 2 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 50°C and 2 min at 72°C, and finally 10 min at 72°C. PCR products were run and visualized on an ethidium bromide infused 1.5% agarose gel. PCR products were purified and sequenced using Macrogen's Eco-Seq service. Problematic sequences were re-amplified and sequenced using a higher annealing temperature of 60°C to try to eliminate fungal contaminates amplifying instead of the parasite DNA.

Parasite identification

Parasite sequences were first grouped together based on similarity, with sequences that were 99% identical being placed together. Next, the United States National Center for Biotechnology Information ‘MOLE-BLAST Neighbor Search Tool’ was used to find the closest matching reference sequences on the GenBank database (Altschul et al., Reference Altschul, Gish, Miller, Myers and Lipman1990; Benson et al., Reference Benson, Cavanaugh, Clark, Karsch-Mizrachi, Lipman, Ostell and Sayers2013). This tool creates an alignment and a neighbour-joining tree to show the relationship that the query sequence has to the reference sequences in the GenBank non-redundant proteins database. Next, a secondary analysis was performed by placing our sequences within an alignment with all of the relevant closest matching GenBank reference sequences. This allowed us to create a maximum likelihood (ML) tree to see relationships between our sequences and the references taken from GenBank. The sequences were aligned in Seaview v5.0.5 (Gouy et al., Reference Gouy, Tannier, Comte and Parsons2021) using the Muscle algorithm, with conserved sites being selected using the Gblocks program (Castresana, Reference Castresana2000). The phylogenetic trees were constructed using the ML method, using a general time reversible model incorporating gamma correction (GTR+Γ) in PhyML v3.1 (Guindon et al., Reference Guindon, Dufayard, Lefort, Anisimova, Hordijk and Gascuel2010), with bootstrap analysis undertaken using 1000 replicates.

GenBank accession numbers

The DNA sequences generated in this study are available in GenBank accession numbers OP626191 – OP626254 (online supplementary table 3).

Results

Infection rates

Of the 800 gastropods collected, 581 were slugs (Agriolimacidae, Arionidae, Boettgerillidae, Limacidae and Milacidae) and 219 were snails (Discidae, Helicidae, Hygromiidae and Oxychilidae). The most common slug species found were Deroceras invadens (15%), Tandonia budapestensis (13%), Deroceras reticulatum (13%), Arion hortensis (10%), Ambigolimax valentianus (8%), Limacus maculatus (7%), Arion vulgaris (7%), Tandonia sowerbyi (6%), Arion ater (6%), Arion subfuscus (4%), Arion rufus (3%), Arion silvaticus (2%), Limacus flavus (2%), Ambigolimax nyctelius (1%), Limax maximus (1%), Milax gagates (<1%) and Boettgerilla pallens (<1%). The most common snail species found were Cepaea nemoralis (28%), Cornu aspersum (25%), Cepaea hortensis (20%), Trochulus striolatus (10%), Oxychilus alliarius (7%), Monacha cantiana (5%), Discus rontundas (3%), Trochulus hispidus (1%) and Arianta arbustorum (1%).

Overall, 227 specimens were infected (28%) with nematodes or trematodes (or both). Of those, 163 were slugs (28%) and 64 were snails (29%) (table 2; fig. 2). The only gastropod species without any recorded infections were A. arbustorum, B. pallens, D. rotundatus and T. hispidus. Nematodes were found in all other gastropods, with T. budapestensis, D. invadens, C. aspersum, D. reticulatum, A. ater and C. nemoralis accounting for over half of all infections. A total of 533 nematodes were recorded from 190 infected specimens (145 slugs and 45 snails). Of those, only 12 juvenile nematodes were found in 12 hosts (eight slugs and four snails) (table 2). Trematodes were rarer than nematodes, with A. ater, A. hortensis, A. nyctelius, A. rufus, A. silvaticus, A. subfuscus, A. vulgaris, L. flavus, L. maximus and O. alliarius having no recorded trematode infections. A total of 242 trematodes were recorded from 55 specimens (30 slugs and 25 snails) (table 2). Lastly, co-infections of both nematodes and trematodes were even rarer, with only 18 specimens being recorded as co-infected (13 slugs and five snails) (table 2).

Table 2. Gastropods collected and details of number of nematode and trematode (metacercariae) infections.

Note: Gastropod species with zero infections are greyed out. ‘Both’ means a co-infection of nematodes and trematodes within a single specimen.

Fig. 2. Map of collection sites (n = 16) across the city of Nottingham and surrounding areas showing infection rates at each collection site. White = uninfected, grey = nematode infection, dark grey = trematode infection and black = nematode/trematode co-infection (Google 2022).

Of the 16 sites surveyed, infection was found at all of them (table 3). The highest recorded rate of infection was 46% at site 7 (Attenborough Nature Reserve, Nottinghamshire) and site 13 (Edwalton, Nottinghamshire). The lowest recorded rate of infection was 12% at site 5 (Beeston, Nottinghamshire). Nematode infections were found at all 16 sites, with trematode infections found at 13 of the 16 sites (fig. 2). Specimens infected with both nematodes and trematodes were found at nine of the 16 sites.

Table 3. Infection rate of collected gastropods (n = 50) at each site across the city of Nottingham and surrounding areas.

Nematode and trematode identifications

A total of 35 (23 adults, 12 juveniles) nematodes (online supplementary table 1) and 29 trematodes (online supplementary table 2) were genotyped. All sequences were grouped together based on similarity (>99%) and those groups were then matched with their closest GenBank references using the Basic Local Alignment Search Tool and MOLE-BLAST tool (ranked by lowest E-value). The nematode sequences fitted into seven groups, with all groups except group C2 having a GenBank reference match greater than 99% (table 4). The trematode sequences fitted into four groups, with all groups except group F1 having a GenBank reference match greater than 99% (table 4).

Table 4. BLAST-MOLE results (ranked by E-value) for grouped nematode (groups A–D) and trematode (groups E–F) sequences with their top five closest references.

Note: (J) indicates it was a juvenile nematode. Each of the different designated groupings of ITS (nematode) and 18S (trematode) sequences are less than 1% different. Nematode and trematode groups with less than 99% GenBank reference match are coloured grey.

Next, ML trees were created for the nematode and trematode sequences by placing each group together with a range of related GenBank references. The majority of the groups were identified at the species level (fig. 3). Only groups C2 and F1 were not identifiable at the species level. Group C2 was outside of the Cosmocerca/Cosmocercoides genera (fig. 3C) and group F1 was outside of the Opisthioglyphe/Macroderoides/Brachycoelium/Mesocoelium/Auridistomum/Telorchis genera, respectively (fig. 3F).

Fig. 3. Maximum likelihood phylogenetic trees of different nematode (trees A–D) and trematode (trees E–F) species using the ITS and 18S rRNA gene, respectively. Tree A was created using 325 base pairs (bp) of the ITS and is rooted on Amphibiophilus mooiensis. Tree B was created using 306 bp of the ITS and is rooted on A. mooiensis. Tree C was created using 402 bp of the ITS and is rooted on Paraspidodera uncinate. Tree D was created using 409 bp of the ITS and is rooted on A. mooiensis. Tree E was created using 450 bp of the 18S rRNA and is rooted on Michajlovia turdi. Tree F was created using 456 bp of the 18S rRNA and is rooted on Brachycladium goliath. All trees were generated using PhyML v3.1; the numbers on the branches indicate the bootstrap percentages for 1000 replicates (bootstrap values under 50% are not shown). The scale bar represents percentage sequence divergence. Differing alignment lengths are due to the limited length of GenBank references. Accession numbers for all sequences can be found in online supplementary table 3.

Discussion

Rate of infection

The vast majority of gastropods collected and examined were slugs (73%), of which five families were represented (Agriolimacidae, Arionidae, Boettgerillidae, Limacidae and Milacidae). The remaining gastropods were snails, of which four families were represented (Discidae, Helicidae, Hygromiidae and Oxychilidae). The largest families represented were the Arionidae (24%), Agriolimacidae (20%), Helicidae (20%), Milacidae (16%), Limacidae (13%), Hygromiidae (4%), Oxychilidae (2%), Discidae (<1%) and Boettgerillidae (<1%). The overall rate of infections for the gastropods collected was 28%. Both slugs (28%) and snails (29%) had a similar rate of infection. No lungworm species of medical or veterinary importance were found within the city of Nottingham. However, of the 26 gastropod species found, 16 are potential hosts for An. vasorum, eight are potential hosts for Crenosoma vulpis and five are potential hosts for Ae. abstrusus (online supplementary table 4).

Nematodes

A total of 533 nematodes were isolated, with only 12 being juveniles. Juvenile nematodes are a useful indication for the possible presence of lungworm (metastrongyloid) species of veterinary importance such as An. vasorum. Of those 12 juvenile nematodes, no lungworm species were found. Instead, four of them were identified as Angiostoma margaretae (Angiostomatidae), a parasite whose definitive host has been reported to be a milacid slug species (Ross et al., Reference Ross, Haukeland, Hatteland and Ivanova2017). We also found it inside D. invadens (Agriolimacidae) and A. valentianus (Limacidae). Four were identified as an unknown Cosmocercidae species, a family of parasitic nematodes whose definitive hosts are reptiles and amphibians (Baker, Reference Baker1984). Two were identified as Phasmarhabditis hermaphrodita and two were identified as Phasmarhabditis neopapillosa (Rhabditidae). Phasmarhabditis is a genus of facultative parasitic nematodes that can parasitize a broad range of gastropod species (Andrus & Rae, Reference Andrus and Rae2019). Of the adult nematodes identified, all were of non-medical (or veterinary) relevance, belonging to four of the seven gastropod-related nematode families (Agfidae, Angiostomatidae, Cosmocercidae and Rhabditidae).

The interactions these nematode families have with terrestrial gastropods are poorly understood (Wilson & Grewal, Reference Wilson, Grewal, PS, R-U and DI2005). The most understood species is P. hermaphrodita, which has been developed into an effective biological alternative molluscicide (Nemaslug®) that reduces agricultural damage done by gastropod pests (Rae et al., Reference Rae, Verdun, Grewal, Robertson and Wilson2007). Unlike chemical molluscicides, Nemaslug® has no adverse effects on non-target organisms such as beneficial organisms (acarids, annelids, carabids, collembolans, dipterans, isopods and nematodes), or gastropod predators (amphibians, birds, mammals and reptiles) (Iglesias et al., Reference Iglesias, Castillejo and Castro2003). However, unlike chemical molluscicides, Nemaslug® cannot kill every gastropod pest species. This is due to P. hermaphrodita only being able to kill smaller gastropod species (e.g. Deroceras spp. and Arion hortensis) and the juveniles of some larger species (Arion ater and Cornu aspersum) (Rae, Reference Rae2017), while larger gastropod species (Ambigolimax spp., Cepaea hortensis, Limacus spp., Limax spp. and Lissachatina fulica) are resistant to the fatal effects of P. hermaphrodita (Williams & Rae, Reference Williams and Rae2015; Rae, Reference Rae2017).

Trematodes

A total of 242 trematodes were isolated. Of these, 29 were genotyped, 14 were identified as Brachylaima arcuata, 11 were identified as B. fuscata and three were identified as B. mesostoma. All these Brachylaima species are common gastrointestinal parasites of the bird families Corvidae, Sylviidae and Turdidae (Heneberg et al., Reference Heneberg, Sitko and Bizos2016). One other trematode sample (belonging to group F1) could not be identified at the species-level. It clustered closely with the genera Opisthioglyphe, Macroderoides, Brachycoelium, Mesocoelium, Auridistomum and Telorchis, placing it within the Plagiorchioidea superfamily. Genera of this Plagiorchioidea superfamily are common parasites of amphibians, fishes and reptiles (Tkach et al., Reference Tkach, Snyder and Swiderski2001).

Brachylaima is a common gastrointestinal parasite of birds, mammals, and reptiles. There are over 60 described species, with Brachylaima being found in Africa, the Americas, Asia, Europe, and Oceania (Nasir & Rodriguez, Reference Nasir and Rodriguez1966; Wheeler et al., Reference Wheeler, Roberts, Beverley-Burton and Sutton1989; Richards et al., Reference Richards, Harris and Lewis1995; Awharitoma et al., Reference Awharitoma, Okaka and Obaze2003; Butcher & Grove, Reference Butcher and Grove2005; Richardson & Campo, Reference Richardson and Campo2005; Gállego et al., Reference Gállego, González-Moreno and Gracenea2014; Gracenea & Gállego, Reference Gracenea and Gállego2017; Nakao et al., Reference Nakao, Waki, Sasaki, Anders, Koga and Asakawa2017; Gérard et al., Reference Gérard, Ansart, Decanter, Martin and Dahirel2020; Termizi & Him, Reference Termizi and Him2021). Brachylaima cribbi is the only documented species capable of infecting humans (Butcher & Grove, Reference Butcher and Grove2001) with brachylaimiasis first documented in 1996, with 13 more cases in the subsequent decades after its discovery, all occurring in Australia (Butcher et al., Reference Butcher, Talbot, Norton, Kirk, Cribb, Forsyth, Knight and Cameron1996; Gállego & Gracenea, Reference Gállego and Gracenea2015). Brachylaimiasis causes diarrhoea, abdominal pain, anorexia, eosinophilia and weight loss (or decreased weight gain) in infected humans, with a predicted mortality rate of 5–10% in untreated patients (Gállego & Gracenea, Reference Gállego and Gracenea2015). Transmission is typically from either the consumption of undercooked land snails (such as Cornu aspersum) infected with metacercariae, or the unintentional consumption of infected gastropod slime/faeces/corpse-contaminated fruits and vegetables (Butcher & Grove, Reference Butcher and Grove2001).

While the consumption of snails is unpopular in the United Kingdom, on average the world consumes 450,000 tonnes (496,040 US tons) of edible snails every year, of which only 15% come from snail farms (López et al., Reference López, Recabal and Carrasco2015). Spain, France, Portugal and Belgium are the biggest importers of snails, with approximately 17 million kilograms of snails being imported as a whole from 2020–2021 (United Nations, 2022). Concerns about the rates of Brachylaima infection in C. aspersum at farms and markets has already been raised in France and Spain (Gállego & Gracenea, Reference Gállego and Gracenea2015; Gracenea & Gállego, Reference Gracenea and Gállego2017; Gérard et al., Reference Gérard, Ansart, Decanter, Martin and Dahirel2020). It is unknown what effect non-B. cribbi species have on public health as there are no studies exploring the possibility of brachylaimiasis caused by European Brachylaima species. Furthermore, brachylaimiasis could be frequently misdiagnosed or overlooked in Europe due to either a lack of experience in identifying it or due to how small Brachylaima eggs are in human faeces (<30 μm in length) (Gracenea & Gállego, Reference Gracenea and Gállego2017).

Supplementary material

To view supplementary material for this article, please visit https://doi.org/10.1017/S0022149X22000645.

Funding

None.

Conflicts of interest

None.

Ethical statement

None.

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Figure 0

Table 1. Collection sites surveyed across the city of Nottingham and surrounding areas.

Figure 1

Fig. 1. Map of collection sites (n = 16) across the city of Nottingham and surrounding areas (Google 2022).

Figure 2

Table 2. Gastropods collected and details of number of nematode and trematode (metacercariae) infections.

Figure 3

Fig. 2. Map of collection sites (n = 16) across the city of Nottingham and surrounding areas showing infection rates at each collection site. White = uninfected, grey = nematode infection, dark grey = trematode infection and black = nematode/trematode co-infection (Google 2022).

Figure 4

Table 3. Infection rate of collected gastropods (n = 50) at each site across the city of Nottingham and surrounding areas.

Figure 5

Table 4. BLAST-MOLE results (ranked by E-value) for grouped nematode (groups A–D) and trematode (groups E–F) sequences with their top five closest references.

Figure 6

Fig. 3. Maximum likelihood phylogenetic trees of different nematode (trees A–D) and trematode (trees E–F) species using the ITS and 18S rRNA gene, respectively. Tree A was created using 325 base pairs (bp) of the ITS and is rooted on Amphibiophilus mooiensis. Tree B was created using 306 bp of the ITS and is rooted on A. mooiensis. Tree C was created using 402 bp of the ITS and is rooted on Paraspidodera uncinate. Tree D was created using 409 bp of the ITS and is rooted on A. mooiensis. Tree E was created using 450 bp of the 18S rRNA and is rooted on Michajlovia turdi. Tree F was created using 456 bp of the 18S rRNA and is rooted on Brachycladium goliath. All trees were generated using PhyML v3.1; the numbers on the branches indicate the bootstrap percentages for 1000 replicates (bootstrap values under 50% are not shown). The scale bar represents percentage sequence divergence. Differing alignment lengths are due to the limited length of GenBank references. Accession numbers for all sequences can be found in online supplementary table 3.

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