Bacterial endosymbionts, in genera Wolbachia and Cardinium, infect various arthropods and some nematode groups. Manipulating these microbial symbionts presents a promising biocontrol strategy for managing disease-causing parasites. However, the diversity of Wolbachia and Cardinium in nematodes remains unclear. This study employed a genome skimming strategy to uncover their occurrence in plant-parasitic nematodes, analyzing 52 populations of 12 species. A metagenome analysis revealed varying endosymbiont genome content, leading to the categorization of strong, weak, and no evidence for endosymbiont genomes. Strong evidence for Wolbachia was found in five populations, and for Cardinium in one population, suggesting a limited occurrence. Strong Wolbachia evidence was noted in Pratylenchus penetrans and Radopholus similis from North/South America and Africa. Heterodera glycines from North America showed strong Cardinium evidence. Weak genomic evidence for Wolbachia was observed in Globodera pallida, Meloidogyne incognita, Rotylenchus reniformis, Pratylechus coffeae, Pratylenchus neglectus, and Pratylenchus thornei; for Cardinium was found in G. pallida, R. reniformis and P. neglectus; 27/52 populations exhibited no endosymbiont evidence. Wolbachia and Cardinium presence varied within nematode species, suggesting non-obligate mutualism. Wolbachia and Cardinium genomes differed among nematode species, indicating potential species-specific functionality. This study advances knowledge of plant-parasitic nematode–bacteria symbiosis, providing insights for downstream eco-friendly biocontrol strategies.

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.

Urbanization has been previously shown to affect evolution of plants and animals by restricting gene flow, increasing genetic drift, and causing divergent selection between rural and urban populations, however, the effect of urbanization on terrestrial gastropod evolution has only been minimally researched. Most research on terrestrial gastropods primarily focusing on invasive pest species, and little attention is given to native species like Ariolimax columbianus (Pacific banana slug). There is no current published research on the phylogeography of A. columbianus. In this study, surface epithelial cells from 66 Ariolimax columbianus individuals were collected from 11 locations around Corvallis, Oregon, and one site in northern California. These samples were amplified and sequenced at two loci: cytochrome oxidase 1 (CO1) and the internal transcribed spacer 2 (ITS-2). This data was used to generate maximum-likelihood phylogenetic trees to better understand the population genetics of these local native gastropods. The CO1 phylogenetic analysis showed two major haplotypes around the city of Corvallis – northwestern and southern, while the ITS-2 phylogenetic analysis showed three general haplotypes around the city – northern, western, and southern. The results in this study show the presence of genetic breaks between many of these populations, possibly due to a combination of factors including anthropogenic fragmentation, natural barriers, and the low active dispersal ability of A. columbianus . However, the extent that each of those factors has on disrupting gene flow and explaining patterns of genetic variation in this species requires further research. The present study acts as a foundation for the development of future research on banana slug phylogenetics and population genetic structure.

Slugs are cryptic terrestrial pests that target a wide range of crops and are especially damaging to seedlings. Management of these invertebrates mostly relies on synthetic chemistry. These molecules can be efficient against slugs and snails but can be toxic to other organisms (e.g., dogs) and harmful to the environment (e.g., leaching into surface and groundwater). The usage of pathogenic nematodes has been effective in several crops and European countries. A survey was conducted to investigate the presence of natural populations of malacopathogenic nematodes in soybean in the mid-Atlantic region. Slugs were sampled in nine fields across Delaware at various distances from the field edges (0 m, 5 m, 10 m, 15 m). Soil cover and soil type were also recorded. Invertebrates were brought back to the laboratory. Slugs were monitored for four weeks, and mortality was classified into one of three categories: (1) death with the presence of nematodes; (2) death with the presence of fungi; (3) death without the presence of nematodes or fungi. Nematodes associated with slugs were identified based on 18S rRNA sequencing. The distance from the field edge did not impact the number of trapped slugs and the incidence of slug death associated with the presence of nematodes. Overall, nematodes were collected from ca. 20% of the slug cadavers, and most have previously been associated with slugs (ca. 35% of deaths associated with fungi and ca. 45% not associated with nematodes or fungi). The number of captured slugs and slug death associated with the presence of nematodes were positively correlated with ground cover. Soil type impacted both the number of captured slugs and the presence of pathogenic nematodes. This survey provides a first insight into the natural populations of mollusk-associated nematodes in the mid-Atlantic region. This knowledge may contribute to implementing cultural practices favoring these natural enemies of slug pests.

The facultative-parasitic nematode Phasmarhabditis hermaphrodita has been used for many years as a biological control agent targeting slug pests. During the nematode’s infection process, the presence of certain bacteria has been suggested to affect the pathogenicity and efficiency of the nematodes in killing slugs, though the potential roles of different bacteria in affecting host-infection by nematodes remain unclear. In this study, we examined three experimental P. hermaphrodita populations cultured with three different bacteria: 1) Escherichia coli (EC), 2) a newly isolated Pseudomonas sp. strain (PS) that co-cultured with a P. hermaphrodita strain found in Oregon, USA, and 3) the original complex bacterial community (BC) associated with the nematode. For each treatment, we evaluated the pathogenicity of P. hermaphrodita at low and high concentrations towards adult grey field slug Deroceras reticulatum and investigated changes in the nematode microbiome structure before and after slug infection. Slugs exposed to EC, of both low and high concentration treatments, survived significantly longer than slugs exposed to PS high and BC high. Slugs in the BC low treatment survived significantly longer compared to BC high, but significantly shorter compared to EC high. We identified a wide variety of taxa components (82 genera) in the community associated with the nematode pre-infection, most of which were of low abundance. In all bacterial treatments post-infection, the number of genera almost quadrupled and the abundance of these taxa changed remarkably, although the taxa with the highest abundance remained stable. We also observed four Pseudomonas amplicon sequence variants (ASVs) that increased in abundance after slug infection in the Pseudomonas treatment. This finding suggests these taxa may play a role in the infection process, potentially acting as opportunistic pathogens, or facilitating infection progression through providing nematodes with a favorable food source, or contributing directly to the nematode’s virulence.