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.

Listening to Slugs: Acceptability and Consumption of Molluscicide Pellets by the Grey Field Slug, Deroceras reticulatum.

Gastropod damage to crop plants has a significant economic impact on agricultural and horticultural industries worldwide, with the Grey Field Slug (Deroceras reticulatum (Müller)) considered the main mollusc pest in the United Kingdom and in many other temperate areas. The prevailing form of crop protection is pellets containing the active ingredient, metaldehyde. Metaldehyde can cause paralysis and death in the mollusc, depending on the amount ingested. The paralysing effects may result in reduced pellet consumption. A greater understanding of metaldehyde consumption may reveal an area that can be manipulated using novel molluscicide formulations. Novel pellet types included commercial metaldehyde pellets coated so that metaldehyde is released more slowly. In both laboratory and arena trials, an audio sensor was used to record individual slugs feeding on a variety of pellet types, including commercially available toxic pellets (metaldehyde and ferric phosphate) and novel metaldehyde formulations. The sensor was used to record the length of each bite and the total number of bites. There was no significant difference in the length of bites between pellet types in laboratory trials. Novel pellets were not consumed more than commercial pellet types. Commercial pellet types did not differ in consumption.

Stability of Patches of Higher Population Density within the Heterogenous Distribution of the Gray Field Slug Deroceras reticulatum in Arable Fields in the UK.

Exploitation of heterogenous distributions of Deroceras reticulatum, in arable fields by targeting molluscicide applications toward areas with higher slug densities, relies on these patches displaying sufficient spatio-temporal stability. Regular sampling of slug activity/distribution was undertaken using 1 ha rectangular grids of 100 refuge traps established in 22 commercial arable field crops. Activity varied significantly between the three years of the study, and the degree of aggregation (Taylor's Power Law) was higher in fields with higher mean trap catches. Hot spot analysis detected statistically significant spatial clusters in all fields, and in 162 of the 167 individual assessment visits. The five assessment visits in which no clusters were detected coincided with low slug activity (≤0.07 per trap). Generalized Linear Models showed significant spatial stability of patches in 11 fields, with non-significant fields also characterized by low slug activity (≤1.2 per trap). Mantel's permutation tests revealed a high degree of correlation between location of individual patches between sampling dates. It was concluded that patches of higher slug density were spatio-temporally stable, but detection using surface refuge traps (which rely on slug activity on the soil surface) was less reliable when adverse environmental conditions resulted in slugs retreating into the upper soil horizons.

Not All Slugs Are the Same: Variation in Growth and Development of the Slug Deroceras reticulatum.

Models to forecast slug populations make assumptions about growth and mortality in response to environmental factors. To refine these models, the growth trajectories and survival of Deroceras reticulatum, a worldwide pest, hatching in spring and autumn were compared at three rearing temperatures (ambient, 12 °C and 15 °C). Deroceras reticulatum reared under identical conditions showed great variation in growth and strong bimodality in growth rates. At all rearing temperatures, growth was influenced by hatching season; in all cases, fast growers dominated in autumn and slow growers dominated in spring. Survival was influenced by hatching season: autumn-born slugs survived better at ambient temperatures, but spring-born slugs had better survival at 15 °C. Deroceras reticulatum may be partitioned into "slow growers" and "fast growers". Fast growers responded to warmer conditions, growing to large sizes. Slow growers, in contrast, gained weight at comparable rates to ambient reared slugs, regardless of the elevated constant temperatures. The peaks of slug activity in autumn and spring are possibly not distinct generations as some slugs may mature early/late and slip into the alternative cohort. Rather, the observed autumn and spring peaks in slug numbers may be a response of a mixed-age population to the favourable environmental conditions at that time.

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.

Nematodes associated with terrestrial slugs in the Edmonton region of Alberta, Canada.

A survey of nematodes associated with terrestrial slugs was conducted in residential gardens, nurseries, greenhouses and agricultural sites located in and around Edmonton, Alberta, Canada. A total of 2406 slugs were collected from 82 sites. Slugs were decapitated and cadavers were incubated for two weeks, with emerging nematodes removed and processed for identification. Nematodes were identified using molecular sequence data for the 18S ribosomal DNA. Nematodes were recovered from 20 of the 82 sites surveyed, with 24.4% of the slugs infected with nematodes. A total of seven nematodes were identified to species level, including Caenorhabditis elegans, Panagrolaimus papillosus, Pellioditis typica, Pelodera pseudoteres, Rhabditella axei, Rhabditoides inermiformis and Phasmarhabditis californica. An additional four specimens were identified to genus level, including Oscheius sp. (9), Pristionchus sp., Rhabditis sp. and Rhabditophanes sp. (1). The two most common nematode species were C. elegans and P. pseudoteres. The facultative parasite, P. californica, was recovered from a single Arion rufus specimen, collected from a seasonal nursery. To our knowledge, this study represents the first survey of slug-associated nematodes in Canada.

The mitochondrial genome of Tetrahymena rostrata.

Tetrahymena rostrata is a ciliated protist which can parasitize the gray field slug, Deroceras reticulatum. Here, we report the sequence of the mitochondrial genome (mt genome) of a strain of T. rostrata that was isolated from the egg of D. reticulatum. Whole cell genomic DNA was sequenced using Illumina® MiSeq and the mitochondrial DNA sequence reads were extracted and assembled. The resulting 47,235 bp assembly contained rRNAs, tRNAs, and 45 protein coding DNA sequences of which 21 encoded proteins of unknown function. Phylogenetic analysis showed T. rostrata clustered with Tetrahymena thermophila, Tetrahymena pigmentosa, Tetrahymena pyriformis, Tetrahymena paravorax, and Tetrahymena malaccensis.

Prevalence of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Crenosoma vulpis larvae in native slug populations in Germany.

Metastrongyloid parasites represent sparsely studied parasites of dogs and cats in Germany. Recent European surveys indicate that these parasites are spreading in Europe. Actual data on prevalence of Angiostrongylus vasorum in dogs and foxes reveal several endemic foci in Germany. However, actual data on the prevalence of A. vasorum and other metastrongyloid lungworm larvae in a wide range of slug and snail intermediate hosts, such as Arion lusitanicus, are missing for Germany. To fill this gap, we conducted an epidemiological survey on native German slugs in selected regions of Hesse and Rhineland-Palatinate. The focus was on slugs, because in study areas slugs appear to be more abundant than snails. Slugs were collected throughout different seasons of the year in areas that were previously proven to be hyperendemic for A. vasorum fox infections. Overall, a total of 2701 slugs were collected and examined for lungworm larvae via artificial digestion. The number of A. vasorum larvae per slug varied considerably (1-546 larvae per specimen). Some hotspot areas with high A. vasorum prevalence in slugs (up to 19.4%) were identified. The overall A. vasorum prevalence varied with season with largest number of slugs infected in summer (9.1%) and lowest number in winter (0.8%). The current study revealed a total A. vasorum prevalence of 4.7% in slugs based on microscopic analyses. Confirmation of lungworm species was made by specific duplex-real-time PCRs. Hence, these data demonstrate that final hosts are at a permanent risk for A. vasorum infections during all seasons when living in investigated areas. Besides A. vasorum, other lungworm larvae were also detected, such as Crenosoma vulpis (the fox lungworm, 2.3%) and Aelurostrongylus abstrusus (feline lungworm, 0.2%).

Neuropeptides predicted from the transcriptome analysis of the gray garden slug Deroceras reticulatum.

The gray garden slug, Deroceras reticulatum (Gastropoda: Pulmonata), is one of the most common terrestrial molluscs. Research for this slug has focused mainly on its ecology, biology, and management due to the severe damage it causes on a wide range of vegetables and field crops. However, little is known about neuropeptides and hormonal signalings. This study, therefore, aimed to establish the transcriptome of D. reticulatum and to identify a comprehensive repertoire of neuropeptides in this slug. Illumina high-throughput sequencing of the whole body transcriptome of D. reticulatum generated a total of 5.9 billion raw paired-end reads. De novo assembly by Trinity resulted in 143,575 transcripts and further filtration selected 120,553 unigenes. Gene Ontology (GO) terms were assigned to 30,588 unigenes, composed of biological processes (36.9%), cellular components (30.2%) and molecular functions (32.9%). Functional annotation by BLASTx revealed 39,987 unigenes with hits, which were further categorized into important functional groups based on sequence abundance. Neuropeptides, ion channels, ribosomal proteins, G protein-coupled receptors, detoxification, immunity and cytoskeleton-related sequences were dominant among the transcripts. BLAST searches and PCR amplification were used to identify 65 putative neuropeptide precursor genes from the D. reticulatum transcriptome, which include achatin, AKH, allatostatin A, B and C, allatotropin, APGWamide, CCAP, cerebrin, conopressin, cysteine-knot protein hormones (bursicon alpha/beta and GPA2/GPB5), elevenin, FCAP, FFamide, FVamide (enterin, fulicin, MIP and PRQFVamide), GGNG, GnRH, insulin, NdWFamide, NKY, PKYMDT, PRXamide (myomodulin, pleurin and sCAP), RFamide (CCK/SK, FMRFamide, FxRIamide, LFRFamide, luqin and NPF), and tachykinin. Over 330 putative peptides were encoded by these precursors. Comparative analysis among different molluscan species clearly revealed that, while D. reticulatum neuropeptide sequences are conserved in Mollusca, there are also some unique features distinct from other members of this species. This is the first transcriptome-wide report of neuropeptides in terrestrial slugs. Our results provide comprehensive transcriptome data of the gray garden slug, with a more detailed focus on the rich repertoire of putative neuropeptide sequences, laying the foundation for molecular studies in this terrestrial slug pest.

Performance of the slug parasitic nematode Phasmarhabditis hermaphrodita under predicted conditions of winter warming.

The nematode Phasmarhabditis hermaphrodita is the only commercial biological control agent for terrestrial slugs. We investigated whether the predicted conditions of winter warming could have any effect on its performance. In the presence of nematodes, slug damage to lettuce plants and slug survival were significantly lower under the predicted conditions of winter warming than under normal winter conditions, while in the absence of nematodes, slug damage and survival were similar under the conditions of winter warming and under current winter conditions. The data suggest that P. hermaphrodita may perform better under the predicted conditions of winter warming.

The complete mitochondrial genome of the gray garden slug Deroceras reticulatum (Gastropoda: Pulmonata: Stylommatophora).

The complete mitochondrial genome sequence of Deroceras reticulatum has been sequenced and annotated in this study. The mitogenome of D. reticulatum is 14,048 base pairs in length, and contains 13 protein-coding genes (PCGs), 22 transfer RNA genes, and 2 ribosomal RNA genes. The overall base composition is 31.0% A, 12.2% C, 17.7% G, and 39.1% T. Based on phylogenetic analysis using the amino acid sequences of PCGs, D. reticulatum was shown to be closely related to other species of Stylommatophora. The first mitochondrial genome from the Agrolimacidae family provides valuable molecular data for taxonomical identification and further evolutionary studies of terrestrial slugs.

Relationships Between Aphids (Insecta: Homoptera: Aphididae) and Slugs (Gastropoda: Stylommatophora: Agriolimacidae) Pests of Legumes (Fabaceae: Lupinus).

Lupin plants are frequently damaged by various herbivorous invertebrates. Significant among these are slugs and aphids, which sometimes attack the same plants. Relationships between aphids, slugs and food plant are very interesting. Grazing by these pests on young plants can lead to significant yield losses. There is evidence that the alkaloids present in some lupin plants may reduce grazing by slugs, aphids and other invertebrates. In laboratory study was analyzed the relationships between aphid Aphis craccivora and slug Deroceras reticulatum pests of legumes Lupinus angustifolius. It was found that the presence of aphids significantly reduced slug grazing on the plants. The lupin cultivars with high alkaloid content were found to be less heavily damaged by D. reticulatum, and the development of A. craccivora was found to be inhibited on such plants.

The effect of climate manipulations on the herbivory of the pest slug Deroceras reticulatum (Müller, 1774) (Pulmonata: Agriolimacidae).

The pestiferous status of the terrestrial slug Deroceras reticulatum and the strong dependence of its biology and ecology on climatic factors have driven research on the potential responses of the slug to predicted scenarios of climate change. Here, we report two short-term experiments performed outdoors, under seminatural conditions, to assess the behavioural response of D. reticulatum to different climate manipulations in terms of herbivory, by measuring over 7 days the damage inflicted by slug populations to lettuce seedlings. The climate manipulations tested emulate predicted climatic conditions for northwest Spain, specifically winter warming and increased summer rainfall, in contrast respectively with normal winter conditions and summer without rain conditions. In a winter experiment, we compared a normal winter treatment with a winter warming treatment; with respect to the normal winter treatment, the winter warming treatment was characterised by higher temperature, lower relative humidity and the absence of rainfall. In a summer experiment, we compared a summer drought treatment with an increased summer rainfall treatment; with respect to the summer drought treatment, the increased summer rainfall treatment was characterised by the presence of rainfall, while the conditions of temperature and relative humidity were similar in both treatments. Neither winter warming nor increased summer rainfall did lead to a significant increase on the number of seedlings damaged by the slugs. However, with both treatments, we found a moderate increase on the amount of damage suffered by the seedlings. The results are discussed in the context of the potential responses of D. reticulatum to future climatic conditions.

The acceptability of meadow plants to the slug Deroceras reticulatum and implications for grassland restoration.

BACKGROUND AND AIMS: Despite the selective pressure slugs may exert on seedling recruitment there is a lack of information in this context within grassland restoration studies. Selective grazing is influenced by interspecific differences in acceptability. As part of a larger study of how slug-seedling interactions may influence upland hay meadow restoration, an assessment of relative acceptability is made for seedlings of meadow plants to the slug, Deroceras reticulatum. METHODS: Slug feeding damage to seedling monocultures of 23 meadow species and Brassica napus was assessed in microcosms over 14 d. The severity and rate of damage incurred by each plant species was analysed with a generalized additive mixed model. Plant species were then ranked for their relative acceptability. KEY RESULTS: Interspecific variation in relative acceptability suggested seedlings of meadow species form a hierarchy of acceptability to D. reticulatum. The four most acceptable species were Achillea millefolium and the grasses Holcus lanatus, Poa trivialis and Festuca rubra. Trifolium pratense was acceptable to D. reticulatum and was the second highest ranking forb species. The most unacceptable species were mainly forbs associated with the target grassland, and included Geranium sylvaticum, Rumex acetosa, Leontodon hispidus and the grass Anthoxanthum odoratum. A strong positive correlation was found for mean cumulative feeding damage and cumulative seedling mortality at day 14. CONCLUSIONS: Highly unacceptable species to D. reticulatum are unlikely to be selectively grazed by slugs during the seedling recruitment phase, and were predominantly target restoration species. Seedlings of highly acceptable species may be less likely to survive slug herbivory and contribute to seedling recruitment at restoration sites. Selective slug herbivory, influenced by acceptability, may influence community-level processes if seedling recruitment and establishment of key functional species, such as T. pratense is reduced.

Cryptosporidium parvum en gastrópodos silvestres como bioindicadores de contaminación fecal en ecosistemas terrestres / Cryptosporidium parvum in wild gastropods as bioindicators of fecal contamination in terrestrial ecosystems

Cryptosporidium sp oocysts were detected in snails (Helix aspersa Milller) and slug (Deroceras reticulatum Milller) from the Valparaiso Region, Chile. Snails and slug were collected from public squares and private domestic gardens. Cryptosporidium sp oocysts were recovered from faeces of both species. Ziehl Neelsen stain, nested PCR, and sequencing analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that snails and slug could act as a reservoir and mechanic vector of C. parvum infection for humans and animals. Moreover, gastropods could serve as bioindicators of fecal soil contamination.

Se detectó ooquistes de Cryptosporidium sp en caracoles (Helix aspersa Müller) y babosas (Deroce-ras reticulatum Müller) de la Región de Valparaíso, Chile. La recolección de caracoles y babosas se efectuó en parques públicos y jardines de diferentes hogares. Los ooquistes de Cryptosporidium sp fueron recuperados de las deposiciones de ambas especies. Después de la tinción de Ziehl Neelsen y la RPC anidada, el análisis de secuenciación demostró un patrón similar a lo descrito para el genotipo C o 2 del parásito. Estos resultados demuestran que los caracoles y babosas podrían actuar como reservorio y vectores mecánicos de la infección por Cryptosporidium. parvum para humanos y animales. Además, estos gastrópodos podrían ser usados como bio-indicadores de contaminación fecal del suelo.

Distribution of Deroceras reticulatum (Müller, 1774) (Pulmonata Stylommatophora) in Argentina with first record of the Reserva de Usos Múltiples Isla Martin Garcia, Río de la Plata superior / Distribução de Deroceras reticulatum (Müller, 1774) (Pulmonata, Stylommatophora) na Argentina com primeiro registro na Reserva de Usos Múltiplos Ilha Martín García, Río de la Plata superior

Deroceras reticulatum is a misanthropic European species spread widely throughout South America. At the moment this species is considered a 'pest' in direct sowing such as maize, soybean, sunflower, wheat, alfalfa and clovers, among others. The aim of this paper is to report the first record of D. reticulatum in the Reserva de Usos Multiples Isla Martín García, Buenos Aires province and to provide information about this species distribution in five Argentina provinces.

Deroceras reticulatum é uma espécie sinantrópica oriunda da Europa e com grande dispersão na América do Sul. Esta espécie é considerada atualmente praga dos cultivos de semeadura direta de milho, soja, trigo, girassol, alfafa e trevos, entre outros. O presente trabalho tem por objetivo comunicar o primeiro achado do agriolimácido D. reticulatum na Reserva de usos múltiplos Ilha Martín García, província de Buenos Aires e proporcionar informação sobre a distribuição desta espécie em cinco províncias argentinas.

Selective grazing of acyanogenic white clover: Variation in behaviour among populations of the slug Deroceras reticulatum.

Collections of the slug Deroceras reticulatum were made from grassland sites containing contrasting frequencies of the cyanogenic morph of white clover, Trifolium repens. In choice chamber experiments, slugs obtained from sites with a low frequency of cyanogenic clover showed a significantly greater degree of selective eating of acyanogenic morphs than slugs taken from a site containing a high frequency of cyanogenic clover. Differences in selectivity between populations were caused both by differences in the rate of initiation of feeding on cyanogenic morphs, and by differences in the extent of damage once feeding had been initiated. The implications of these results for the cyanogenic polymorphism of T. repens are discussed.