The United States harbors a significant diversity of invasive species (Pimentel et al., 2005). They serve as a threat to the country’s natural biodiversity since the introduction of invasive species is one of the leading causes of global biodiversity decline (Mckinney and Lockwood, 1999; Clavero and Garcia-Berthou, 2005; Butchart et al., 2010; Gladstone and Bordeau, 2020). While the distribution of numerous invasives have been tracked, some taxonomic groups have been largely neglected, notably terrestrial gastropod species, many species of which are of agricultural and horticultural interest (Barker, 2002; Pyšek et al., 2008; Lowry et al., 2012; Gladstone and Bordeau, 2020).

Many invasive terrestrial gastropods in the United States are present on the west coast, especially in California, Oregon, Washington, and Hawaii, where most gastropod surveys have been conducted. For example, in California, it is estimated that there are approximately 279 species of terrestrial gastropods, 37 of which are invasive (Roth and Sadeghian, 2003). These gastropods were likely introduced via the horticultural trade when gastropods residing on plant products were delivered to western states (Cowie et al., 2008; Bergey et al., 2014).

Some of these introduced gastropods are considered among the most pestiferous slugs and snails. These species include Deroceras reticulatum (Müller 1774), Arion hortensis (Ferrusac 1819), and Cornu aspersum (Müller 1774) (Mc Donnell et al., 2009). For example, C. aspersum can reduce some California citrus fruit crop yields by 40–50% and occasionally up to 90–100% in years of high rainfall (Pappas and Carman, 1961; Sakovich, 2002). Terrestrial gastropods do not only cause direct physical damage to plants, but they can also spread disease. They have been found to serve as vectors for pathogens like Alternaria brassicicola (Saccardo 1880), and members of the family Peronosporaceae, and other plant-pathogenic fungi (Wester et al., 1964; Hasan and Vago, 1966; Turchetti and Chelazzi, 1984). Some gastropod species have also been found to harbor human pathogens. It has been postulated that some slugs and snails have been partially responsible for spinach and other salad crop recalls due to the discovery of Campylobacter spp. and Escherichia coli (Migula 1895) in the feces of sampled gastropods (Sproston et al., 2006; Raloff, 2007). Multiple terrestrial gastropods have also been found to carry Angiostrongylus cantonensis (Chen 1935), the causative agent for eosinophilic meningitis (Lindo et al., 2004; Teem et al., 2013; Iwanowicz et al., 2015).

Invasive terrestrial gastropods can also be detrimental to sensitive ecosystems. Multiple wetlands and marshes are threatened by invasive gastropod species because they thrive in these environments with a lack of natural predators (Cowie, 1998; Silliman et al., 2005). The invasive gastropods are capable of quick propagation and can reach large populations within a relatively short period of time. These large invasive populations result in a lack of resources for other endemic organisms which cannot compete with the invasives (Cowie, 1998; Silliman et al., 2005). Additionally, some native snail species have disappeared due to the introduction of the carnivorous snail Euglandia rosea (Ferrusac, 1821) (Cowie, 1998; Silliman et al., 2005). Horticultural and agricultural trade across the world brings danger to endemic organisms. To prevent invasive gastropods from being introduced through horticultural and agricultural trade, effective methods of pest control must be utilized.

The most common method of gastropod pest control is the use of molluscicides. One of the most widely used molluscicides is metaldehyde formulated as pelleted baits. These baits attract gastropods and upon ingestion are rapidly hydrolyzed to acetaldehyde, which causes the animal to produce excess mucus, dehydrate, and ultimately die (Triebskorn et al., 1998; Castle et al., 2017). However, these baits have variable efficacy due to a range of factors including weather conditions, different levels of attractiveness, and failure of a gastropod to consume enough bait (Crowell, 1967). Also, metaldehyde baits, along with most other molluscicides, are not targeted methods of pest control. Metaldehyde baits can harm a variety of different organisms including dogs, humans, and other organisms upon consumption (Castle et al., 2017). For example, ranking below chocolate ingestion, metaldehyde poisoning is the second most common cause of poisoning in canines (Cope et al., 2006). In mammals, metaldehyde is an irritant to the skin, eyes, mucous membranes, throat, and respiratory tract (Castle et al., 2017). The active ingredient may be leached at points of application and found in downstream river catchments at a level that can cause harm to non-target populations away from the application site (Gillman et al., 2012).

Parasitic nematodes within the genus Phasmarhabditis can be effective biological control agents against pestiferous gastropods with more targeted results compared to molluscicides (Rae et al., 2007). There are currently 16 nominal species of Phasmarhabditis worldwide (Wilson et al., 1993; Azzam, 2003; Huang et al., 2015; Nermut et al., 2016a,b; Tandingan De Ley et al., 2016; Ivanova and Spiridonov, 2017; Nermut et al., 2017; Pieterse et al., 2017; Ross et al., 2018; Pieterse, 2020; Zhang and Liu, 2020; Ivanova and Spiridonov, 2021). All species tested for their biological control potential have been shown to specifically target and kill gastropods, providing protection to a variety of crops (Wilson et al., 1993; Rae et al., 2007; Mc Donnell et al., 2018b,Mc Donnell et al., 2020; Nermut et al., 2020; Tandingan De Ley et al., 2020). Phasmarhabditis hermaphrodita is the most well-known and well-studied member of the genus. It is a facultative parasite that feeds on bacteria and can live saprobically or necromenically on gastropods or their feces (Tan and Grewal, 2001). P. hermaphrodita has seen success as a biological control agent across Europe as the commercially available product Nemaslug^®. The species has undergone non-target testing with various species of earthworms, as well as native, non-pest European slugs and snails (Wilson et al., 2000; Grewal et al., 2003; Rae et al., 2005; Nardo et al., 2010). It did not cause mortality in any of the non-target species tested, suggesting that it is a safer alternative in Europe to traditional molluscicides, which are lethal to many organisms other than gastropods.

Until recently Phasmarhabditis had not been isolated within the United States. Therefore, due to agricultural policies, such as the National Environmental Policy Act (Montgomery, 2011), the commercialized Eurasian strain was not approved for use within the United States and as of 2021, it is still not commercially available in the United States. To find a viable biological control agent for gastropods in the United States, gastropod-nematode surveys within the country were performed to find a local species of nematode capable of causing mortality in slugs and snails. Three different surveys from 2000 to 2010 were performed to search for a gastropod biological control agent in the United States (Grewal et al., 2000; Kaya and Mitani, 2000; Ross et al., 2010). Most of the surveys looked for the presence of Phasmarhabditis, but they also searched for a variety of other nematode species found within gastropods and assessed their virulence. None of the surveys recovered any Phasmarhabditis species or other candidates for a gastropod biological control agent. However, over the past 8 years three species of Phasmarhabditis have been confirmed in California and one has been found in Oregon (Tandingan De Ley et al., 2014, 2016; Mc Donnell et al., 2018a). Thus, these local populations of Phasmarhabditis species should be the focus of future gastropod biological control research in the United States.

The first series of surveys which lead to the discovery of three Phasmarhabditis species in the United States in 2014 were conducted from 2012 to 2017 in California nurseries and garden centers. They were performed to search for potential biocontrol agents of invasive snails or slugs and to determine the distribution of parasitic nematodes including Phasmarhabditis. The species identified were P. hermaphrodita, P. papillosa, and a newly described species P. californica (Tandingan De Ley et al., 2014, 2016). As the next step, we evaluated the potential use of the local strains as biological control agents against invasive pestiferous gastropods in California (Tandingan De Ley et al., 2020).

Additional surveys were performed in 2018–2021 to determine the presence and distribution of gastropods and their associated Phasmarhabditis species, and to determine if the genus is widely established throughout the state. This series of extensive gastropod-nematode surveys is the first in the state of California. Such surveys have the potential to identify previously unknown nematode-gastropod relationships or identify new species of nematodes with biocontrol potential. These types of discoveries have been seen in other surveys performed across the globe (Ross et al., 2012; Tandingan De Ley et al., 2014; Mc Donnell et al., 2018a; Brophy et al., 2020). In this survey, we aimed to determine the presence and distribution of Phasmarhabditis nematodes and the diversity of gastropods in nurseries and garden centers throughout California.

This was the first extensive gastropod and nematode survey performed throughout California. The surveys from 2012 to 2017 and 2018 to 2020 combined covered a total of 28 counties and resulted in the collection of 18 different gastropod species from 6,590 specimens. A total of 69 Phasmarhabditis isolates were collected. The most common gastropod species recovered was A. valentianus. According to this survey, invasive slug and snail species are more common than native gastropod species in California nurseries. Gastropod abundance decreased as we moved southward through California (Figures 1–3). This could be due to the desert and chapparal climates which occur in most of the southern sections of California, while Northern California climates include more precipitation. Slugs are prone to desiccation; therefore, lower survival rates in these climate conditions are plausible (Sternberg, 2000).

Phasmarhabditis species were found throughout all three geographic areas of California (Northern, Central, and Southern). Three species were identified throughout the state, P. hermaphrodita, P. californica, and P. papillosa. Also, one isolate was recovered in Monterey County which seems to be a close relative to or a variant of P. papillosa (Figure 4); with morphological characteristics diagnostic to the species. However, based on genetic analyses of the D2-D3 expansion segments of rRNA, it varies by 2 transitions, 1 ambiguity, 1 transversion, and 2 insertions/deletions (Thymine instead of Cytosine in nucleotide positions 46 and 67; C/T instead of Cytosine in position 140, Adenine instead of a Cytosine in position 261, and 2 indels on positions 346 and 347, respectively). Surveys in Oregon showed the same 3 Phasmarhabditis species, and interestingly, P. hermaphrodita was recovered from a slug in a Brassica field in Salem, OR, suggesting it may also be present in the wider agricultural environment (Howe et al., 2020). In California, our surveys focused solely on nurseries and garden centers because (1) horticulture is one of the most valuable agricultural industries in the state (2) slugs and snails are major pests in the industry and (3) transportation of plants to and from retail nurseries presumably causes gastropods to be moved around the state over great distances than would otherwise be possible. The nurseries themselves could therefore be focal locations for exposure of invasive slug and snail species to a greater diversity of gastropod associated nematodes than is likely to occur in production fields or greenhouses. Based on the Oregon finding, it is likely that these gastropod-infecting nematodes may have also found their way as hitchhikers into agricultural and horticultural fields and backyard gardens in California. However, that has yet to be determined as these production areas were not covered in our surveys.

Additionally, recent studies based on mitochondrial DNA COI gene phylogenies, showed that Phasmarhabditis hermaphrodita U.S. isolates and strains (including isolates from the CA 2012 to 2017 survey and OR surveys) had haplotypes that were nearly identical to P. hermaphrodita collected in the United Kingdom for commercialization of Nemaslug^®. They were placed together in an intraspecific monophyletic clade with the Nemaslug^® strain (Howe et al., 2020). We can hypothesize that P. hermaphrodita found in the United States likely came from areas where Nemaslug^® was used in Europe. Invasion of California and Oregon probably came about from agricultural trade with interstate movement of infected soil and/or slugs/snails. The invasive slugs from Europe and some of the specimens found in this survey likely came with these same nematodes of near-identical haplotypes. It is equally likely that the nematodes came to California on slugs many years ago before the commercialization of Nemaslug^®, and they have stayed in the region by infecting Deroceras slugs and other suitable host pest slugs which are now established in California. In the study by Howe et al. (2020), available P. californica strains at that time were also studied. As with P. hermaphrodita, all P. californica haplotypes (CA, United States; United Kingdom; and New Zealand) belonged to one single, strongly supported clade. Interestingly, P. californica shares the same geographical niche and host gastropod species as P. hermaphrodita from Northern to Southern California. However, P. papillosa seems to only inhabit areas of Southern California (Figure 4).

Some gastropod species were more commonly infected with Phasmarhabditis than others. The gastropod host A. valentianus, which was the most frequently found gastropod, only accounted for about 8.5% of the Phasmarhabditis isolates collected. A. valentianus may have a more developed immune response to parasitic nematodes compared to other slugs, however, this has yet to be determined. The majority of Phasmarhabditis nematodes collected from the survey were collected from D. reticulatum. The host D. reticulatum accounted for about 55% of all Phasmarhabditis nematodes collected. In total, the genus Deroceras accounted for about 74.1% of the identified Phasmarhabditis nematodes (Table 2). D. reticulatum is a common slug pest across Europe, especially in areas near Ireland and the United Kingdom where Nemaslug^® was originally discovered (Kerney, 1999). This serves as additional evidence that an infected invasive species of gastropod from Europe likely brought Phasmarhabditis to the United States where the relationship between the gastropod hosts remained.

Multiple gastropods collected throughout the survey were infected and/or associated with a diverse array of nematodes (other than Phasmarhabditis) (Supplementary Table 1 and Tables 3, 4). C. elegans and C. remanei were the most prominent nematodes found within gastropod hosts. These nematodes are not uncommon in gastropods, and though the interaction between C. remanei and gastropods has not been thoroughly explored, C. elegans is thought to have a phoretic association with gastropods (Caswell-Chen et al., 2005; Ross et al., 2012; Petersen et al., 2015; Rae, 2017; Sudhaus, 2018). C. elegans is also known to have phoretic associations with some species of earthworms and arthropods (Kiontke and Sudhause, 2006; Brophy et al., 2020). Other interesting nematode species were also discovered during the California surveys including Cosmocercoides tonkinensis, which is not commonly associated with gastropod hosts (Supplementary Table 1 and Tables 3, 4; Sudhaus, 2018). C. tonkinensis has only been described in reptiles (Tran et al., 2015). However, for another member of the genus, Cosmocercoides dukae, mollusks are a known host (Anderson, 1960). A survey done in 2014 which identified P. hermaphrodita in California also recovered species other than Phasmarhabditis within gastropod hosts. Some of these species included Alloionema appendiculatum (a common parasite of slugs), C. elegans, C. briggsae, a new species A. similis, and species of Oscheius (Tandingan De Ley et al., 2014; Holovachov et al., 2016). Our non-Phasmarhabditis results share in some of these genus recoveries, except for Alloionema spp. (Table 3). The absence of this genus throughout both surveys spanning from 2012 to 2021 is unexpected and intriguing since it was discovered in past surveys in similar locations (Laznik et al., 2010).

The occurrence of P. hermaphrodita and other species of the genus in North America has regulatory implications for potential biocontrol strategies against non-native slug and snail species that are pests of agriculture on this continent. Since the nematode occurs throughout the state, its use in a similar manner to Nemaslug^® may be a feasible option. Its use could potentially save the California specialty crop industry about 64 million dollars, and is therefore worth exploring as a biological control option (Supplementary Table 2). The recovery of Phasmarhabditis from local plant nurseries and garden centers throughout California was not entirely surprising as these are considered transport hubs for non-native gastropod species (Bergey et al., 2014). It is not known if Phasmarhabditis exists in the natural environment throughout California where horticultural practices do not take place. In order to better understand the presence of Phasmarhabditis in the state, further surveillance is required in horticultural and agricultural field production areas, as well as natural ecosystems. Also, additional non-target and target host experiments with Phasmarhabditis are required to have a deeper understanding of how these potential biological control agents will affect the local ecosystem where they would likely be introduced. Additionally, host experimentation should be performed in mesocosms or other field-like conditions to determine efficacy.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Mendeley Data, V2, doi: 10.17632/dwxh3bmmcb..

JS carried out nematode studies and identification, conceptualized, planned, and carried out the survey, identified gastropods, maintained gastropods and nematodes and created records for all specimens, performed nematode genetic identification work, and wrote and designed the manuscript. ITD conceptualized, planned, carried out, and supervised the nematode genetic studies and identification, secured fundings, performed the survey for gastropods and nematodes throughout California, and assisted in the writing and development of the manuscript. KA assisted in genetic identification and maintenance of gastropods and nematodes. TP provided guidance and secured funding throughout early stages of the project. RM performed survey for all gastropods throughout California between 2012 and 2017, assisted in gastropod identification during the surveys between 2018 and 2020, secured funding, and assisted in the development of the manuscript. AD secured funding, managed the project, and assisted in the development of the manuscript. All authors contributed to the article and approved the submitted version.

ITD, RM, and TP, declare that they are co-inventors on a patent entitled Mollusk-killing Biopesticide (WO2017059342A1). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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