Slugs are a persistent threat to Maryland soybean, typically infesting < 20% of soybean acreage but causing significant yield loss when populations reach high densities (Musser et al. 2018, 2019). The sporadic-but-severe nature of slug damage makes management frustrating. Ironically, insecticides make slug problems worse by killing predators but leaving slugs unharmed (Douglas et al. 2015). Molluscicides (e.g., metaldehyde or iron phosphate, applied as a bait) can be effective, but are too costly and prone to washing away with rain to be relied upon as a preventative treatment (Bailey 2002). By the time slug damage is evident, though, it may already be too late to achieve control with a molluscicide. This is a classic “damned if you do, damned if you don’t” conundrum. Moving forward, we need to understand what factors put a soybean field at greater risk of economic damage by slugs so that we can manage our farms to avoid situations
where stand loss becomes unacceptably high.

Natural predators and parasites (enemies) of slugs are a perhaps underappreciated ally in our battle against slugs. A variety of ground beetles, spiders, marsh flies, and nematodes are known to consume slugs at different parts of the slug life cycle (Barker 2004). These natural enemies are themselves influenced by a number of factors, including weather, tillage, pesticide use, and cover crops (Everts et al. 1989; Vernavá et al. 2004; Le Gall & Tooker 2017; Rivers et al. 2018). Sometimes, the link between natural enemy numbers and slug numbers appears clear. For example, a common ground beetle (Pterostichus melinarius) readily consumed a common slug (Deroceras reticulatum) in a small grains farm in the UK, and slug numbers dropped with increasing beetle numbers (Symondson et al. 2002). However, it is unclear how farms can leverage these natural enemies.

The Maryland Grain Producers Utilization Board and the Delaware Soybean Board provided support for Dr. Crossley shortly after his arrival at University of Delaware to begin examining the natural enemies that could make a dent in slug populations and the factors that promote these natural enemies. Dr. Crossley, along with help from Dr. David Owens and the effort of a dedicated PhD student (Thabu Mugala), were able to sample >1,000 slugs (~3% of which harbored slug parasitic nematodes) and >1,000 ground beetles that are currently being identified to species before using molecular techniques to confirm which species are important slug predators. At least two types of slug parasitic nematodes were collected (with obvious morphological differences), one type was from marsh slugs, the other from leopard slugs. We propose to continue this line of research, and will specifically focus in 2023 on finding more slug parasitic nematodes and developing a method for maintaining nematodes in liquid culture (as opposed to continually feeding them living slugs), which will greatly enhance our ability to identify and manipulate them for slug pathogenicity experiments.

For this project, I propose to: 1) Collect and identify slug parasitic nematodes from Maryland soybean fields, and 2) Develop a liquid culturing technique to maintain slug parasitic nematode colonies in the lab.

1) Collect and identify slug parasitic nematodes from Maryland soybean fields.
2) Develop a liquid culturing technique to maintain slug parasitic nematode colonies in the lab.

Objective 1: We will continue sampling slugs and natural enemies using shingle traps and pitfall traps placed within no-till soybean fields in the Spring of 2023 to collect and identify ground beetles and slug parasitic nematodes. We learned last spring that the number of slugs caught under shingle traps could be improved by placing a cup of water in a hole beneath the shingle trap. Because we observed a low rate of nematode infection (~3%) in 2022, we will use this modification to increase the number of slugs caught to boost our sample size of nematodeinfected slugs.

Importantly, we are always looking for collaborating farms that are willing to let us sample slugs and natural enemies from their farms. Please reach out to Mike if interested (crossley@udel.edu).

To collect slug parasitic nematodes, all captured slugs will be reared in the lab under ideal conditions for 3 weeks, at which point infected slugs will melt and nematodes will emerge seeking new hosts. Melted slugs will be placed in white traps (basically a petri dish with water to attract dispersing nematodes). A subsample of nematodes will be used for morphological identification and DNA sequencing (using the COI barcode) to confirm species identity.

Objective 2: We ultimately aim to test the pathogenicity of any slug parasitic nematodes that we collect, with the goal of developing a highly effective strain of nematodes that can be used for slug control. A critical step, after identification, is to buildup nematode populations under optimal conditions in the lab. Typically, nematodes are kept on living slugs and then
transferred to new slugs once their host slugs melt. This is an especially laborious process, and would require thousands of slugs to be kept in the lab to buildup nematode populations. Here, we propose to develop a liquid culture technique to maintain and buildup nematode populations. It is indeed possible to grow nematodes without slug hosts because nematodes primarily feed on bacteria (not the actual slug host). Without going into great detail on the protocol, the approach is essentially to grow the bacteria that nematodes feed upon in a soup of liquid bread (oil, salt, sugar, etc.). We would like to develop this approach to maintain slug parasitic nematode colonies in the lab for eventual experiments to determine slug pathogenicity.

Update:
Objective 1. A total of 17 fields were regularly sampled, yielding a total of 1,531 slugs (1,043 marsh slugs, 488 gray garden slugs). Only 20 marsh slugs (~2%) melted and produced nematodes, a low but typical proportion. These slugs originated in just two sites. No gray garden slugs were infected with nematodes. Identification is ongoing, but so far we have identified two of the nematode species. One of them, Panagrolaimus detritophagus, is not considered a strict parasite, but instead uses slug hosts to disperse and feeds on bacteria in the host and environment. The other, Pristionchus pacificus, is suspected to be a true parasite based on one of the few studies on the species in natural environments. We plan to eventually conduct pathogenicity tests to verify the potential of this nematode to serve as an effective slug parasite.
Objective 2. We have developed the process for producing the substrate used to grow the bacteria that feed the nematodes. Efforts are currently underway to isolate and culture these bacteria from slugs.

Update:
Objective 1: Collect and identify slug parasitic nematodes from Maryland soybean fields.

A total of 17 fields were regularly sampled, yielding a total of 1,531 slugs (1,043 marsh slugs, 488 gray garden slugs). Only 20 marsh slugs (~2%) melted and produced nematodes, a low but typical proportion (~3% of slugs melted in 2022). These slugs originated in just two sites. No gray garden slugs were infected with nematodes. Identification is ongoing, but so far we have identified three of the nematode species. One of them, Panagrolaimus detritophagus, is not considered a strict parasite, but instead uses slug hosts to disperse and feeds on bacteria in the host and environment. The other, Pristionchus pacificus, is an obscure species that has only been recorded parasitizing scarab beetles. Finding it in a slug is exciting, but warrants further investigation to determine pathogenicity. The third species was identified to the genus Oscheius, which includes species that are known slug parasites. We are most excited about finding this nematode. We plan to continue identifying these nematodes, and to eventually conduct pathogenicity tests to verify the potential of these nematodes to serve as effective slug parasites.

Objective 2: Develop a liquid culturing technique to maintain slug parasitic nematode colonies in the lab.

Work on this front is ongoing. Nematodes are currently being maintained in colonies that are occasionally replenished by infecting live slugs and collecting emergent nematodes. However, the goal is to be able to maintain nematodes without slugs and to ramp up numbers to be able to do pathogenicity tests. To do so, we have begun isolating bacteria from slugs and culturing them on agar petri dishes. The next step is to identify these bacteria, and then get them into liquid culture to feed nematode colonies. Toward this end, we just acquired an incubated orbital shaker, a necessary piece of equipment to maintain these bacterial cultures (they need to be kept cool and aerated by continuous shaking). This equipment was purchased using my own lab startup funds. We plan to continue this work to the point of being ready for pathogenicity testing.

We succeeded in collecting 20 nematode strains from marsh slugs on the Delmarva peninsula, and have so far identified 3 species (identification work is ongoing). Two of these species are potentially slug parasites, while the other is known to occasionally use slugs as a means of transportation (transportation options are limited, I guess). Work to develop a technique to culture nematodes in the lab is ongoing. We have secured necessary equipment (using lab funds) and chemicals, and have begun isolating bacteria that will serve as a food source for nematodes kept in liquid culture.

In 2021, Maryland farms planted 490,000 acres of soybean, averaging 53 bu/acre with a value > $311 million (USDA NASS 2021). Slugs represent a severe, albeit sporadic pest of soybean, for which the costs of treating or not treating can be equally costly. Often, by the time stand loss is evident, the window of opportunity for rescue treatment is long gone. Knowledge about the natural enemy complex that interacts with slugs and of the factors that promote natural suppression of slugs is needed to support an integrated management approach for slugs in lowtill soybean.