Promoting natural suppression of slugs using local parasitic nematodes

Progress report for GNE22-294

Project Type: Graduate Student
Funds awarded in 2022: $15,000.00
Projected End Date: 11/10/2024
Grant Recipient: University of Delaware
Region: Northeast
State: Delaware
Graduate Student:
Faculty Advisor:
Dr. Michael Crossley
University of Delaware
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Project Information


As more farms in the US Northeast adopt conservation agriculture practices such as reduced tillage and cover cropping, new challenges to pest management continue to emerge, reinforcing the axiom that “no good deed goes unpunished”. Slugs, particularly the gray garden slug (Deroceras reticulatum) and the marsh slug (Deroceras leave) have emerged as prominent pests of corn and soybean under no-tillage systems in the United States. Chemical control using molluscicide baits is the most common control method. However, these baits are expensive to apply, ineffective in many field conditions, and harmful to humans and wildlife. Biological control represents an important alternative approach to slug management for growers practicing conservation agriculture. Slug-parasitic nematodes can be potent biological control agents, and one species is already being deployed in Europe. However, slug-parasitic nematodes are so far unknown and unutilized in the US Northeast. This is because there are only a few “locally” isolated strains in the United States (from the West Coast) and importation of such species is highly restricted. Therefore, the proposed research aims to: 1) Identify species of slug-parasitic nematodes and their prevalence in no-till corn and soybean production systems in Delaware and Maryland, and 2) Determine the effectiveness (% mortality induced, impact of infection on slug feeding behavior) of these slug-parasitic nematodes against slugs. This represents an important step toward developing an effective and sustainable slug management strategy for Northeastern grain production and the project will provide significant training for the student committed to promoting integrated pest management in the USNortheast.

Project Objectives:

Objective 1: Identify species of slug-parasitic nematodes and their prevalence in no-till corn and soybean production systems in Delaware and Maryland

Objective 2: Determine the effectiveness of slug-parasitic nematodes against gray garden slugs and marsh slugs:

2a) Determine percent slug mortality caused by different slug parasitic nematode species using different concentrations (lethal doses) under laboratory conditions

2b) Define the amount of time it takes for nematode-infected slugs to stop feeding


The purpose of this project is to determine environmentally and ecologically sound management options for gray garden slugs and marsh slugs using locally isolated slug-parasitic nematodes, reducing dependency on chemical molluscicide baits without sacrificing pest control. Slugs, especially gray garden slug and marsh slug, have emerged as major pests in corn and soybean production systems under conservation practices (Douglas & Tooker, 2012). Both species feed on a wide variety of cultivated and non-cultivated plants in addition to cover crop residues (Gall & Tooker, 2017).  In no-till corn and soybean, slug damage is severe in the early growing season, usually when juvenile slugs feed on seeds and emerged seedlings (Dively & Patton, 2022).   Severe damage has been reported when planting date aligns with slug hatch, which exposes seeds, seedlings, or young plants to a rapidly growing slug population (Douglas & Tooker, 2012).

Slug damage is associated with leaf shredding in corn and destroyed cotyledons or seedling death in soybean  (Brichler, 2020). High levels of injury cause yield reduction and in some cases require replanting of entire fields  (Douglas & Tooker, 2012). Damage is most prominent in young plants because older plants can outgrow slug injury (Abendroth, et al., 2009; Douglas & Tooker, 2012). The control of slugs through the use of chemical molluscicides applied in granular baits is difficult because the baits are easily thwarted by rain, have a short field life-span, and need to be applied in close synchrony with pest activity (Henderson & Triebskornz, 2002; Castle, et al., 2017). Thus, there remains a need for effective and environmentally sound slug management strategies.

Slug-parasitic nematodes represent a powerful potential biological control agent because they inhabit the soil, actively locate cryptic hosts in their environment, and harbor symbiotic bacteria with potent molluscicidal properties (Tan & Grewal, 2001). Even though the Western US (California, Oregon) has reported isolation of local strains of a slug-parasitic nematode species ( (Mc Donnell, De Ley, & Paine, 2018; Mc Donnell, Colton, Howe, & Denver, 2020), the Northeast has yet to isolate and record presence of any species or strains. In Europe, one slug-parasitic nematode species is already commercialized, but importation of nematodes is greatly restricted in the US (Mc Donnell, De Ley, & Paine, 2018; Mc Donnell, Colton, Howe, & Denver, 2020). Furthermore, slug-parasitic nematodes can be highly species-specific, with most control failures following from the use of unsuitable nematode strains or unfavorable environmental conditions  (Rae, Robertson, & Wilson, 2009). Slug-parasitic nematodes thus represent uniquely adapted biological control agents whose efficacy must be examined and optimized within the environmental and geographical context where management is needed. This project will identify nematode species present in the US Northeast and their efficacy against the two economically important slugs in this region, and represents an important step toward alleviating the apparent trade-off between conservation agriculture and effective slug management.


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Materials and methods:

Objective 1: Identify species of slug-parasitic nematodes and their prevalence in no-till corn and soybean production systems in Delaware and Maryland

Origin of slugs and nematodes

  • Marsh and gray garden slugs will be collected from 10 different corn and soybean fields (Sites) in Delaware and Maryland, during the spring of 2022, 2023, and 2024. Slugs will be collected using shingle traps and carried to the laboratory using plastic containers with perforated lids and lined with moist paper towels. Shingle traps will be placed along two transects for a total of 8 traps per field, sampled weekly from March-July for a two-year period (Figure 1). In the laboratory, slugs will be fed carrots and leafy vegetables, replaced on a daily basis, and the containers containing slugs will be cleaned weekly and slug mortality recorded.
  • Project workflow

Figure 1. Slug-parasitic nematode isolation and identification workflow.

Isolation, Morphological and Molecular identification

Slug-parasitic nematode isolation involved putting slug cadavers on petri dishes and then transferring them onto modified white traps to encourage reproduction (Pieterse, Tiedt, Malan, & Ross, 2017). A White trap is a nematode trapping technique that uses nematodes’ attraction to water and successfully recovers the infective stages of the nematodes (Figure 1)  (Orozco, Lee, & Stock, 2014). The modified White trap by Kaya and Stock (1997) consists of a petri dish, on which the host cadaver rests, placed in another larger petri dish containing water. As infective juveniles emerge, they move to the surrounding water trap where they are harvested, and the process can be expanded to commercial production levels  (Shapiro-llan & Hall, 2012) .Infective juveniles will be harvested within the first week of emergence, after which storage will be through the use of culturing flasks held at 14°C- 19°C in the laboratory and re-cultured on a freeze-killed slugs every 4 weeks. Slug parasitic nematodes needed for molecular analysis will be stored in 100% ethanol, while for morphological analysis, the slug-parasitic nematodes will be killed and fixed with hot TAF (2% triethanolamine, 8% formalin in distilled water)  (Pieterse, Tiedt, Malan, & Ross, 2017). Morphological identification of slug parasitic nematodes will be through mounted microscope slides with a paraffin wax ring and the slides containing nematodes will be viewed and the nematodes measured using a compound microscope with a fitted camera and live measurement capacity (Leica DM200, Leica Microsystems). For molecular identification, nematodes will be identified by sequencing cytochrome C oxidase 1 (COI) (Figure 1). First, DNA will be isolated from nematodes using a DNeasy Blood and Tissue Kit (Qiagen). Second, a portion of the COI gene will be PCR-amplified using a combination of universal primers following  (Mahmoud, et al., 2019). Third, COI amplicons will be submitted for Sanger sequencing at the University of Delaware DNA Sequencing & Genotyping Center. Lastly, resulting sequences will be compared to a global database of nematode COI sequences using NCBI BLAST.

Research results and discussion:

I collected 1,030 slugs in 2022 and 1,531 slugs in 2023 from 14 corn and soybean fields in Delaware and Maryland. Among these slugs, four species were represented:  leopard slug (Limax maximus), three-banded slug (Ambigolimax valentianus), marsh slug (Deroceras laeve), and gray garden slug (Deroceras reticulatum). The collected slugs in 2022 mainly consisted of marsh slugs (858), followed by gray garden slugs (121), leopard slugs (27), and three-banded slugs (24). In 2023, I only observed marsh slugs (1,043) and gray garden slugs (488). In total, only 30 of the collected slugs were parasitized by nematodes (3%) in 2022, and 60 slugs were parasitized by nematodes (4%) in 2023. These nematodes are currently being maintained in liquid cultures in my laboratory (Fig 2). From these nematode isolates I have so far identified  three species  through DNA sequencing, namely Oscheius myriophilus, Panagrolaimus detritophagus, and Pristionchus pacificus. Preliminary slug pathogenicity trials based on these isolates are anticipated to be conducted in the year 2024.

nematode and culture flasks

Fig. 2. A. Magnified slug parasitic nematode and B. Nematode culture flasks on an orbital shaker



Participation Summary
8 Farmers participating in research

Education & Outreach Activities and Participation Summary

1 Webinars / talks / presentations
1 Workshop field days

Participation Summary:

420 Farmers participated
Education/outreach description:

1 presentation: SPARK Symposium at the University of Delaware

I presented on slug parasitic nematodes at the SPARK symposium, a competitive program hosted by the University of Delaware graduate college to feature outstanding graduate research while providing professional communication training to selected speakers. I also won the people's choice award at this event.


Project Outcomes

4 Grants applied for that built upon this project
4 Grants received that built upon this project
$99,196.00 Dollar amount of grants received that built upon this project
6 New working collaborations
Project outcomes:

Our research project will contribute to future sustainability  by identifying local parasitic nematode species that have the potential to provide control of a key early season pest without reliance on costly and environmentally toxic molluscicides. These parasitic nematodes are naturally found in the soil and can actively locate and kill their slug hosts in the field without causing environmental contamination or imbalance. Overall, having a deeper understanding of the mollusk parasitic nematode species present in commercial agricultural fields and their pathogenicity potential is a cardinal step towards developing a sustainable mollusk management program and mollusk parasitic nematode-based control technologies

Knowledge Gained:

Our project's main goal has been working towards effective management of slugs within corn and soybean production systems cultivated under conservation agriculture principles (cover cropping and no-till), by increasing knowledge about the impacts of slug-parasitic nematodes on farms. We have been working on our first objective, which aims to isolate parasitic slug nematodes from the fields in the Northeastern region of the United States.

 Our preliminary results have confirmed the presence of slug parasitic nematodes (albeit at low prevalence) and at least three nematode species. One of these species (Panagrolaimus detritophagus) is likely non-parasitic, instead feeding on bacteria in the environment and occasionally using slugs to disperse. Another species (Pristionchus pacificus) has been documented parasitizing beetles, so its emergence from a slug host is intriguing and warrants further study. The last species (Oscheius myriophilus) is a generalist parasite known to affect insects as well as slugs. The future direction for this project is to confirm species identity of the remaining isolates through both molecular and morphological techniques and conduct laboratory and greenhouse trials to determine their pathogenicity toward slugs, while continuing to collect more nematode strains for further testing. We also plan to communicate our findings through journal publications, conference presentations, workshops, and field days.

 I am passionate about promoting sustainable agriculture in rural communities. Thus, the knowledge from this project will help me understand alternative pest management approaches and promote them among small-scale farmers. Professionally, this project will enhance my skills in DNA extraction and sequencing, mass rearing of slug parasitic nematodes using in-vitro techniques, and increasing my visibility and experience through publications. The latter will prepare me for my career, whether I pursue a career in academia or industry.


Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.