Immune-Related Gene Profiles and Differential Expression in the Grey Garden Slug Deroceras reticulatum Infected with the Parasitic Nematode Phasmarhabditis hermaphrodita.

The grey garden slug (Deroceras reticulatum), a common terrestrial slug native to Europe with a global distribution including North America, is commonly considered the most severe slug pest in agriculture. The nematode Phasmarhabditis hermaphrodita, which has been used in the U.K. and Europe as a commercial biocontrol agent since 1994, has also recently been collected in Oregon and California and has long been considered a candidate biocontrol agent for slug management in the U.S. In this study, we report differential gene expressions in nematode-infected slugs using RNA-seq to identify slug immune-related genes against nematodes. Comparison of gene expression levels between the whole bodies of a nematode-infected slug (N-S) and an uninfected control slug (C-S) revealed that there were a total of 39,380 regulated unigenes, of which 3084 (3%) were upregulated and 6761 (6%) were downregulated at greater than 2-fold change (FC > 2) in the nematode-infected slug. To further investigate the biological functions of differentially expressed genes (DEGs), gene ontology (GO) and functional enrichment analysis were performed to map the DEGs to terms in the GO, eukaryotic ortholog groups of proteins (KOG) and Kyoto Encyclopedia of Genes and Genome Pathway (KEGG) databases. Among these DEGs, approximately 228 genes associated with immunity or immune-related pathways were upregulated 2-fold or more in the N-S compared to C-S. These genes include toll, Imd, JNK, scavenger receptors (SCRs), C-type lectins (CTLs), immunoglobulin-like domains, and JAK/STAT63 signaling pathways. From the RNA-seq results, we selected 18 genes and confirmed their expression levels by qRT-PCR. Our findings provide insights into the immune response of slugs during nematode infection. These studies provide fundamental information that will be valuable for the development of new methods of pest slug control using pathogenic nematodes in the field.

The biocontrol nematode Phasmarhabditis hermaphrodita infects and increases mortality of Monadenia fidelis, a non-target terrestrial gastropod species endemic to the Pacific Northwest of North America, in laboratory conditions.

Inundative biological control (biocontrol) efforts in pest management lead to the mass distribution of commercialized biocontrol agents. Many 'biocontrol gone awry' incidents have resulted in disastrous biodiversity impacts, leading to increased scrutiny of biocontrol efforts. The nematode Phasmarhabditis hermaphrodita is sold as a biocontrol agent on three continents and targets pest gastropods such as Deroceras reticulatum, the Grey Field Slug; P. hermaphrodita is not presently approved for use in the United States. Investigations into the potential for P. hermaphrodita to infect non-target gastropod species of conservation relevance, however, are limited. We examined the effects of three strains of P. hermaphrodita on mortality in Monadenia fidelis, the Pacific Sideband, a snail species endemic to the Pacific Northwest of North America, in laboratory conditions. Across a 71-day laboratory infectivity assay, snails exposed to each of the three nematode strains, each analyzed at two doses, experienced a mean 50% mortality by days 20-42. All nematode-treated snails were dead by the end of the study. By contrast, 30/30 water-control snails experienced no mortality. Nematodes killed smaller, juvenile-stage snails significantly faster than those in larger and more developmentally advanced hosts. Our results provide direct evidence that the biocontrol nematode P. hermaphrodita infects and kills M. fidelis, a non-target gastropod species endemic to the Pacific Northwest, in laboratory conditions. This study suggests that introduction of P. hermaphrodita to new ecosystems might negatively impact endemic gastropod biodiversity and advocates for further investigation of non-target effects, including in conditions closer to the natural environments of non-target species.

First Report of the Gastropod-Killing Nematode, Phasmarhabditis californica, in Washington State, U.S.A.

Phasmarhabditis californica, a commercially available biological control agent in England, Scotland, and Wales (Nemaslug 2.0 ®), was discovered for the first time in Washington State during 2022. Nematodes were recovered from the invasive gastropods Arion hortensis, Deroceras reticulatum, and Oxychilus sp. in garden centers in both Vancouver and Spokane. The 18S ribosomal RNA gene was used to identify the nematodes. This discovery builds upon previous reports of P. californica in California and Oregon and suggests that the species is widespread throughout the west coast of the U.S. Future research directions with P. californica are proposed.

Distribution of Phasmarhabditis (Nematode: Rhabditidae) and Their Gastropod Hosts in California Plant Nurseries and Garden Centers.

Three species of Phasmarhabditis were recovered from 75 nurseries and garden centers in 28 counties in California during fall and winter 2012-2021. A total of 18 mollusk species were recovered, most of them invasive. Nematodes were identified by sequencing the D2-D3 expansion segments of the large subunit (LSU or 28S) rRNA. Based on these surveys, P. californica was the most widespread species (37 isolates, 53.6% recovery); followed by P. hermaphrodita (26 isolates; 37.7% recovery); P. papillosa and a closely related P. papillosa isolate (6 isolates; 8.7% recovery). Nematode isolates were mainly collected from four invasive slugs (Deroceras reticulatum, D. laeve, Arion hortensis agg, Ambigolimax valentianus) and snails (Oxychilus spp. and Discus spp.). Results suggest that P. californica and P. hermaphrodita share an ecological niche in Northern, Central, Coastal, and Southern California, north of Los Angeles County.

Feeding habits and multifunctional classification of soil-associated consumers from protists to vertebrates.

Soil organisms drive major ecosystem functions by mineralising carbon and releasing nutrients during decomposition processes, which supports plant growth, aboveground biodiversity and, ultimately, human nutrition. Soil ecologists often operate with functional groups to infer the effects of individual taxa on ecosystem functions and services. Simultaneous assessment of the functional roles of multiple taxa is possible using food-web reconstructions, but our knowledge of the feeding habits of many taxa is insufficient and often based on limited evidence. Over the last two decades, molecular, biochemical and isotopic tools have improved our understanding of the feeding habits of various soil organisms, yet this knowledge is still to be synthesised into a common functional framework. Here, we provide a comprehensive review of the feeding habits of consumers in soil, including protists, micro-, meso- and macrofauna (invertebrates), and soil-associated vertebrates. We have integrated existing functional group classifications with findings gained with novel methods and compiled an overarching classification across taxa focusing on key universal traits such as food resource preferences, body masses, microhabitat specialisation, protection and hunting mechanisms. Our summary highlights various strands of evidence that many functional groups commonly used in soil ecology and food-web models are feeding on multiple types of food resources. In many cases, omnivory is observed down to the species level of taxonomic resolution, challenging realism of traditional soil food-web models based on distinct resource-based energy channels. Novel methods, such as stable isotope, fatty acid and DNA gut content analyses, have revealed previously hidden facets of trophic relationships of soil consumers, such as food assimilation, multichannel feeding across trophic levels, hidden trophic niche differentiation and the importance of alternative food/prey, as well as energy transfers across ecosystem compartments. Wider adoption of such tools and the development of open interoperable platforms that assemble morphological, ecological and trophic data as traits of soil taxa will enable the refinement and expansion of the multifunctional classification of consumers in soil. The compiled multifunctional classification of soil-associated consumers will serve as a reference for ecologists working with biodiversity changes and biodiversity-ecosystem functioning relationships, making soil food-web research more accessible and reproducible.

Fermenting Bread Dough as a Cheap, Effective, Nontoxic, and Generic Attractant for Pest Snails and Slugs.

Invasive slugs and snails are among the most damaging pests of agriculture in temperate and tropical regions of the world. Control options, however, are limited and there is a heavy reliance on chemical molluscicides of variable efficacy. There is an ongoing need to improve management methods. Here, we show that a simple fermenting bread dough formulation (flour, water, and yeast) was effective in attracting pest mollusk species in laboratory tests, and in multiple replicated field trials in Hawaii, Oregon, and Montana. The dough attracted substantially more terrestrial pest gastropods, including invasive species of major economic importance such as Cornu aspersum, Deroceras reticulatum, Ambigolimax valentianus, Xerolenta obvia, Lissachatina fulica, and Parmarion martensi, than water controls. The dough remained attractive for at least 8 days and was significantly more attractive than a widely used metaldehyde-based bait, Deadline® M-Ps™. Thus, fermenting bread dough represents a nontoxic, generic, and effective tool to aid in managing pest gastropod infestations, either using baited traps or in attract-and-kill approaches. Given its simplicity, low cost, and the ready availability of its ingredients, the dough also has potential to be used in developing countries where access to commercial molluscicide baits is limited by cost.

Identification and functional characterization of the first molluscan neuromedin U receptor in the slug, Deroceras reticulatum.

Neuromedin U (NmU) is a neuropeptide regulating diverse physiological processes. The insect homologs of vertebrate NmU are categorized as PRXamide family peptides due to their conserved C-terminal end. However, NmU homologs have been elusive in Mollusca, the second largest phylum in the animal kingdom. Here we report the first molluscan NmU/PRXamide receptor from the slug, Deroceras reticulatum. Two splicing variants of the receptor gene were functionally expressed and tested for binding with ten endogenous peptides from the slug and some insect PRXamide and vertebrate NmU peptides. Three heptapeptides (QPPLPRYa, QPPVPRYa and AVPRPRIa) triggered significant activation of the receptors, suggesting that they are true ligands for the NmU/PRXamide receptor in the slug. Synthetic peptides with structural modifications at different amino acid positions provided important insights on the core moiety of the active peptides. One receptor variant always exhibited higher binding activity than the other variant. The NmU-encoding genes were highly expressed in the slug brain, while the receptor gene was expressed at lower levels in general with relatively higher expression levels in both the brain and foot. Injection of the bioactive peptides into slugs triggered defensive behavior such as copious mucus secretion and a range of other anomalous behaviors including immobilization, suggesting their role in important physiological functions.

Assessment of the Biological Control Potential of Common Carabid Beetle Species for Autumn- and Winter-Active Pests (Gastropoda, Lepidoptera, Diptera: Tipulidae) in Annual Ryegrass in Western Oregon.

While carabid beetles have been shown to feed on a variety of crop pests, little is known about their species assemblages in US annual ryegrass crops, where invertebrate pests, particularly slugs, lepidopteran larvae and craneflies, incur major financial costs. This study assesses the biological control potential of carabid beetles for autumn- and winter-active pests in annual ryegrass grown for seed by: (a) investigating the spatial and temporal overlap of carabids with key pests; and (b) molecular gut content analysis using qPCR. Introduced Nebria brevicollis was the only common carabid that was active during pest emergence in autumn, with 18.6% and 8.3% of N. brevicollis collected between September and October testing positive for lepidopteran and cranefly DNA, respectively, but only 1.7% testing positive for slug DNA. While pest DNA was also detected in the guts of the other common carabid species-Agonum muelleri, Calosoma cancellatum and Poecilus laetulus-these were active only during spring and summer, when crop damage by pests is less critical. None of the four carabid species was affected by disk tilling and only N. brevicollis was significantly associated with a vegetated field margin. However, as its impact on native ecosystems is unknown, we do not recommend managing for this species.

Phylogenetic evidence for the invasion of a commercialized European Phasmarhabditis hermaphrodita lineage into North America and New Zealand.

Biological control (biocontrol) as a component of pest management strategies reduces reliance on synthetic chemicals, and seemingly offers a natural approach that minimizes environmental impact. However, introducing a new organism to new environments as a classical biocontrol agent can have broad and unanticipated biodiversity effects and conservation consequences. Nematodes are currently used in a variety of commercial biocontrol applications, including the use of Phasmarhabditis hermaphrodita as an agent targeting pest slug and snail species. This species was originally discovered in Germany, and is generally thought to have European origins. P. hermaphrodita is sold under the trade name Nemaslug®, and is available only in European markets. However, this nematode species was discovered in New Zealand and the western United States, though its specific origins remained unclear. In this study, we analyzed 45 nematode strains representing eight different Phasmarhabditis species, collected from nine countries around the world. A segment of nematode mitochondrial DNA (mtDNA) was sequenced and subjected to phylogenetic analyses. Our mtDNA phylogenies were overall consistent with previous analyses based on nuclear ribosomal RNA (rRNA) loci. The recently discovered P. hermaphrodita strains in New Zealand and the United States had mtDNA haplotypes nearly identical to that of Nemaslug®, and these were placed together in an intraspecific monophyletic clade with high support in maximum likelihood and Bayesian analyses. We also examined bacteria that co-cultured with the nematode strains isolated in Oregon, USA, by analyzing 16S rRNA sequences. Eight different bacterial genera were found to associate with these nematodes, though Moraxella osloensis, the bacteria species used in the Nemaslug® formulation, was not detected. This study provided evidence that nematodes deriving from the Nemaslug® biocontrol product have invaded countries where its use is prohibited by regulatory agencies and not commercially available.

Development of a High-Throughput Laboratory Bioassay for Testing Potential Attractants for Terrestrial Snails and Slugs.

Invasive snails and slugs are among the most damaging pests of vegetables, fruits, ornamentals, grains, and forage throughout the world. Current control strategies are focused almost exclusively on molluscicides, which are ineffective under some conditions, and which can have serious nontarget effects. A major aim of this study was to develop a generic high-throughput bioassay method for use in identifying attractants for terrestrial gastropods, with the overall goal of developing attractant-based control methods for pest gastropods. Here, we demonstrate the use of the bioassay method in screening a wide variety of foodstuffs and other possible sources of attractants, using the pest snail Cornu aspersum Müller (Pulmonata, Helicidae) and the pest slug Deroceras reticulatum Müller (Pulmonata, Agriolimacidae) as test animals. Among a large number of food items and previously reported attractants tested, chopped fresh cucumber (Cucumis sativus) was the most attractive substrate for both species. Our results also showed that previous feeding experience influences subsequent food choice to some extent, but regardless of previous feeding experience, chopped cucumber was as attractive or more attractive than any other substrate tested.

Alloionema similis n. sp., a genetically divergent sibling species of A. appendiculatum Schneider, 1859 (Rhabditida: Alloionematidae) from invasive slugs in California, USA.

A new species of Alloionema Schneider, 1859, A. similis n. sp., and the known species A. appendiculatum Schneider, 1859 were isolated from cadavers of invasive slugs in California. Both species are described based on morphology, morphometrics and molecular data. Alloionema similis n. sp. is morphologically very similar to A. appendiculatum but can be distinguished by a more posterior position of the excretory pore in the Kleinform females and longer tail in the Kleinform males. Substantial differences between the two species are, however, found in both 18S and 28S rDNA sequences. Sequence analysis revealed unambiguous autapomorphies in nucleotide sequence and secondary structure of rRNA genes, separating A. appendiculatum and A. similis n. sp. Molecular phylogenies were inferred from concatenated secondary-structure based multiple sequence alignments of nearly complete 18S and the D1-D3 domains of the 28S rRNA genes. Phylogenetic analyses placed these two species as sister taxa in a monophyletic clade, separately from Neoalloionema tricaudatum Ivanova, Pham Van Luc & Spiridonov, 2016 and N. indicum Nermut, Puza & Mrácek, 2016.

Assessing metal contamination from construction and demolition (C&D) waste used to infill wetlands: using Deroceras reticulatum (Mollusca: Gastropoda).

Large quantities of construction and demolition waste (C&D) are produced globally every year, with little known about potential environmental impacts. In the present study, the slug, Deroceras reticulatum (Mollusca: Gastropoda) was used as the first biomonitor of metals (Ag, As, Ba, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Sb, Se, Ti, Tl, V and Zn) on wetlands post infilling with construction and demolition (C&D) waste. The bioaccumulation of As, Ba, Cd, Co, Sb, Se and Tl were found to be significantly elevated in slugs collected on C&D waste when compared to unimproved pastures (control sites), while Mo, Se and Sr had significantly higher concentrations in slugs collected on C&D waste when compared to known contaminated sites (mining locations), indicating the potential hazardous nature of C&D waste to biota. Identifying exact sources for these metals within the waste can be problematic, due to its heterogenic nature. Biomonitors are a useful tool for future monitoring and impact studies, facilitating policy makers and regulations in other countries regarding C&D waste infill. In addition, improving separation of C&D waste to allow increased reuse and recycling is likely to be effective in reducing the volume of waste being used as infill, subsequently decreasing potential metal contamination.

Key aspects of the biology of snail-killing Sciomyzidae flies.

The biology of snail-killing flies (Diptera: Sciomyzidae) has been studied intensively over the past half-century, especially over the past decade. Today, sciomyzids are biologically the best-known group of higher Diptera. The overarching research objectives are evaluation of sciomyzids as biocontrols of disease-carrying or agriculturally important snails and slugs and as a paradigm group for the study of the evolution of diverse feeding and associated behaviors in flies. We present reviews and analyses of some key features of particular scientific and societal interest, including behavioral and phenological groups; laboratory experimental studies on behavior and development; population biology, bioindicators, ecosystem service provision, and conservation; phylogenetics, molecular studies, and evolutionary biology; and biocontrol.