Chemotactic Responses of Oscheius myriophilus to Mollusk Mucus

Simple Summary

This study evaluates the potential of the nematode Oscheius myriophilus as a biological control agent for pest mollusks. The researchers analyzed the nematode’s chemotactic response and movement when exposed to the mucus of five mollusk species at two temperatures (20 °C and 25 °C). The results indicated that both the species affiliation of the mollusk and the temperature affected the nematode’s attraction and movement, with higher motility observed at 20 °C. Cernuella virgata mucus was the most effective attractant, especially at the lower temperature. Further studies are recommended to pinpoint the chemical cues in mollusk mucus that drive these behaviors.

Abstract

Terrestrial slugs and snails can significantly harm agriculture. Due to environmental concerns associated with chemical molluscicides, biological control methods are increasingly being explored. Oscheius myriophilus (Poinar, 1986), a nematode species recently discovered in association with Arion vulgaris Moquin-Tandon, 1855, holds promise as a biocontrol agent for gastropod pests. In this study, we investigated the chemotactic response and motility of O. myriophilus when exposed to the mucus of five mollusk species: Helix pomatia Linnaeus, 1758, Cernuella virgata (Da Costa, 1778), Deroceras reticulatum Müller, 1774, A. vulgaris, and Tandonia budapestensis Hazay, 1880. Our experiments were conducted at two temperatures (20 °C and 25 °C) to assess how environmental conditions influence nematode behavior. The results demonstrated that the chemoattractiveness of mollusk mucus to O. myriophilus was significantly influenced by both the species of mollusk and the temperature. Overall, nematode motility was higher at 20 °C than at 25 °C, indicating that lower temperatures may enhance the activity of O. myriophilus. Among the tested mollusk species, C. virgata mucus consistently attracted the highest number of nematodes, especially at the lower temperature. Our findings indicate that the chemotactic response of O. myriophilus to mollusk mucus may have potential for the targeted biocontrol of pest mollusks. While C. virgata demonstrated strong attractant potential at the tested temperatures, particularly under cooler conditions (20 °C), further research is needed to confirm whether this represents a consistent temperature-related effect. Future studies should aim to identify the specific chemical cues in mollusk mucus that trigger nematode attraction and examine how these signals interact with a broader range of environmental variables, including temperature, to influence nematode behavior.

1. Introduction

Gastropods are a diverse group of invertebrates, and out of the approximately 35,000 species of terrestrial slugs and snails, certain species are known to cause significant damage to agricultural crops [1,2]. In Europe, several species stand out as key agricultural pests: D. reticulatum, Arion distinctus Mabille, 1868, Arion hortensis Férussac, 1819, Arion intermedius Normand, 1852, Arion rufus L., 1758, A. vulgaris, C. virgata, and T. budapestensis [1,3,4]. To manage these pests, pellets containing active chemical ingredients such as metaldehyde or iron (III) phosphate are commonly used [5,6,7]. Some molluscicides have been formulated with additional substances to attract slugs and snails, improving the efficiency of the poison as both a food- or contact-based solution [5]. However, chemical molluscicides pose considerable environmental risks to non-target organisms, including earthworms, domestic animals, and humans [8]. Metaldehyde contamination is particularly concerning, as its concentration in surface water often exceeds the European Union’s regulatory threshold of 0.1 µg/L (Directive 98/83/EC; EC, 1998) [9]. Given these toxicological concerns, there is increasing interest in biological control methods as safer alternatives in pest management programs for slugs and snails [10].

The use of molluscicidal nematodes as a biological control method for managing pest gastropods is a promising strategy. Phasmarhabditis Schneider is the only nematode genus that has been commercialized as a biological control agent for pest slugs and snails [11]. Among the 18 known species worldwide [12], two have been developed as commercial products. The first species, Phasmarhabditis hermaphrodita (Schneider, 1859), in combination with its bacterial symbiont Moraxella osloensis Bøvre & Henriksen, 1967 (Moraxellaceae), was introduced as Nemaslug^® in 1994 and has been widely used across Europe for managing gastropod pests in various crops [13]. However, recent research [14] revealed that the bacteria in Nemaslug^® are actually Psychobacter spp., not M. osloensis. The second species, Phasmarhabditis californica Tandingan De Ley, 2016, also formulated with M. osloensis [15], became commercially available in 2022 in England, Scotland, and Wales under the name Nemaslug 2.0^®. Recent studies have highlighted the ability of Phasmarhabditis papillosa (Schneider, 1866) to cause significant mortality in Deroceras panormitanum (Lessona & Pollonera, 1882), D. reticulatum, C. virgata, and A. vulgaris, underscoring its potential as a biological control agent for gastropods, warranting further research [16,17].

The family Rhabditidae consists of free-living, bacterivorous nematodes, with Oscheius being a prominent genus within this group [18]. This genus is categorized into two groups, Insectivora and Dolichura [19], and it encompasses 45 described species [20]. Studies have shown that Oscheius species exhibit entomopathogenic activity, making them useful biological control agents for various invertebrate pests [20,21,22]. In recent years, Oscheius species have gained increasing scientific interest. For example, O. myriophilus has been found associated with garden millipedes in California, millipedes in southern Australia, European mole crickets in Turkey, and sugar cane crop soil in Mexico [22,23,24]. Recent research [17] reported the presence of O. myriophilus in the Spanish slug (A. vulgaris), marking the first recorded instance of this nematode in a gastropod host. This discovery presented an opportunity to explore O. myriophilus’s potential as a biological control agent against various gastropod pests [17].

An effective biocontrol agent must rapidly locate, damage, and kill its host pest, ideally within a timeframe comparable to that of chemical control methods [25]. Parasitic nematodes utilize a range of strategies to identify and infect their hosts. Their motility, direction, and host-finding abilities are influenced by physical and chemical cues present in their environment [26,27,28]. Nematodes can actively pursue signals from food sources or potential hosts, and some even specialize in specific host species or food types [29]. Chemical attractants can significantly reduce the time nematodes need to locate a host, thereby enhancing the efficiency of biocontrol measures against slugs [27]. These chemical signals can affect the speed and frequency of nematode movement [25].

Several studies have investigated the behavioral and chemotactic responses of nematodes such as P. hermaphrodita, P. neopapillosa, P. papillosa, and P. californica to the mucus of various mollusks [7,25,30,31,32,33]. In contrast, the chemotactic behavior of O. myriophila remains underexplored, with current research providing limited data on its interaction with different slug species, especially in response to the chemical cues they emit. To fill this gap in knowledge, we aimed to test whether O. myriophilus exhibits variable chemotactic and motility responses when exposed to the mucus of five distinct mollusk species: the Roman snail (H. pomatia), the common white snail (C. virgata), the gray field slug (D. reticulatum), the Spanish slug (A. vulgaris), and the Budapest keeled slug (T. budapestensis).

2. Materials and Methods

2.1. Mollusk Collection and Maintenance

This study investigated the chemotactic response and motility of the parasitic nematode O. myriophilus in response to mucus from various mollusk species. The experiments were conducted at the Laboratory of Entomology, Biotechnical Faculty, University of Ljubljana, Slovenia, Department of Agronomy. Five agriculturally significant mollusk species in Slovenia were included as the following: the Roman snail (H. pomatia), the common white snail (C. virgata), the gray field slug (D. reticulatum), the Spanish slug (A. vulgaris), and the Budapest keeled slug (T. budapestensis) [17,33].

Mollusks were collected from trial fields at the Biotechnical Faculty in Ljubljana (46°04′ N, 14°31′ E, elevation 299 m) during June and July 2023. The identification of species was performed using established taxonomic guides [4]. Specimens of varying ages were individually housed in aerated plastic containers (6 × 8 × 15 cm) lined with moistened filter paper to prevent desiccation. Each container was provided with 1 mL of water daily. The mollusks were maintained at room temperature (~22 °C) and fed lettuce daily. For calcium supplementation, H. pomatia and C. virgata were also supplied with cuttlebone. To ensure the mollusks were free from natural parasitic infections or diseases, they were kept under these conditions for four weeks before the experiments.

2.2. Nematode Suspensions

Nematodes extracted from slug body cavities were cultured in the laboratory under in vivo conditions using freeze-killed A. vulgaris slugs as a food source, following established protocols [34]. Molecular analysis confirmed their identity as O. myriophilus [17]. After a 10-day cultivation period, nematode cultures were purified by centrifugation with a 5% sodium hypochlorite solution, followed by two distilled water rinses to ensure cleanliness and viability [35].

Infective juveniles (IJs) were then stored in M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 0.5 g NaCl, and 1 g NH₄Cl in 1 L H₂O) at 4 °C, adjusted to a density of 2000 IJs/mL. Only juveniles less than two weeks old with over 95% viability were selected for experiments to ensure high-quality stocks. For additional methodological details, refer to [33].

2.3. Chemotaxis Assay and Mollusk Mucus Collection

To evaluate the chemotactic response of O. myriophilus, a modified method from O’Halloran and Burnell [36] and Jagodič et al. [28] was used. The assay employed 9 cm Petri dishes containing 20 mL of 1.6% agar supplemented with potassium phosphate, CaCl₂, and MgSO₄, divided into three sections: right (treated area), center, and left (control area) [28,33].

In Experiment A, nematode motility was tested using mollusk mucus (50 µL) on the right side and distilled water on the left, with 100 infective juveniles (IJs) placed in the center. Control plates received water on both sides. In Experiment B, nematodes were exposed to mucus from two mollusk species (mollusk A on the right and mollusk B on the left), following the same protocol. Nematodes were counted after 24 h, and the chemotaxis index (CI) was calculated as the following:

CI = (% of IJs in treatment area − % of IJs in control area)/100%

Experiments were conducted at 20 °C and 25 °C with 75% relative humidity in a dark chamber. Plates were frozen at −20 °C to immobilize nematodes before counting. CI values ≥0.20 indicated attraction, and ≤−0.20 indicated repulsion [27,28,33,37].

2.4. Statistical Analysis

Statistical analyses were conducted to evaluate the effects of mucus from four mollusk species and two temperatures on the chemotactic behavior of O. myriophilus. Response variables included nematode motility (percentage of nematodes in the outer vs. inner sections of assay plates) and the chemotaxis index.

In Experiment A, two-way ANOVA analyzed differences in the average number of nematodes migrating to the outer area versus those remaining in the center. In Experiment B, two-way ANOVA assessed the chemotaxis index across different mucus treatments and temperatures. Duncan’s multiple range test (p < 0.05) identified significant differences between means. The results, expressed as the mean ± S.E., are presented in tables and figures. All analyses were performed using Statgraphics Plus for Windows 4.0 (Statistical Graphics Corp., Manugistics, Inc., Rockville, MD, USA).

3. Results

3.1. Nematode Chemoattraction Towards Mollusk Mucus Versus Water

3.1.1. Nematode Motility

The motility of O. myriophilus infective juveniles (IJs) was assessed by tracking their movement from the inner to the outer areas of the assay dish. Nematode motility was significantly influenced by both mollusk mucus (p < 0.01) and temperature (p = 0.02), while temporal (p = 0.52) and spatial repetitions (p = 0.51), as well as the interaction between mollusk mucus and temperature (p = 0.65), did not significantly affect the results (

Table 1. Analysis of variance in the effects of mucus, water, and the temporal and spatial replication of factors on the motility of Oscheius myriophilus.

Factor	Df	F	p
Mollusk mucus	5	10.61	<0.01
Temperature (T)	1	10.43	0.02
Temporal replication	9	1.73	0.52
Spatial replication	2	0.87	0.51
Mollusk mucus × T	5	1.27	0.65
Residual	79
Total (Corrected)	101

).

O. myriophilus exhibited higher activity at the lower temperature of 20 °C compared to 25 °C. On average, 24.63 ± 0.59% of the nematodes moved into the outer area at 20 °C, whereas only 21.41 ± 0.81% were motile at 25 °C, irrespective of treatment type. The highest motility overall was observed in treatments involving mucus from C. virgata, with 35.59 ± 2.07% of nematodes migrating to the outer area, regardless of temperature.

At 20 °C, the highest motility occurred in response to C. virgata mucus (34.16 ± 3.66%), followed by D. reticulatum (31.92 ± 4.83%) and A. vulgaris (29.35 ± 3.80%) (Figure 1). In contrast, the control treatment exhibited the lowest motility, with only 2.15 ± 0.27% of the nematodes moving to the outer area of the assay dish.

At 25 °C, the greatest motility was again observed in response to C. virgata mucus (37.02 ± 2.07%) (Figure 1). No significant differences were found in the motility induced by the mucus of D. reticulatum (23.37 ± 1.49%), A. vulgaris (24.02 ± 6.09%), and T. budapestensis (20.13 ± 2.69%). The control treatment again demonstrated the lowest level of nematode movement, with only 6.70 ± 0.26% migrating to the outer area (Figure 1).

3.1.2. Chemotaxis Index

The chemotaxis index was significantly influenced by the mollusk mucus factor (p < 0.01). Conversely, temperature (p = 0.43), temporal replication (p = 0.63), spatial replication (p = 0.60), and interaction between the mollusk mucus and temperature (p = 0.63) did not significantly affect the chemotaxis index (

Table 2. Analysis of variance in the chemotaxis index.

Factor	Df	F	p
Mollusk mucus	5	7.63	<0.01
Temperature (T)	1	1.78	0.43
Temporal replication	9	0.69	0.63
Spatial replication	2	0.44	0.60
Mollusk mucus × T	5	2.31	0.63
Residual	79
Total (Corrected)	101

).

At a temperature of 20 °C, mucus from A. vulgaris (chemotaxis index = 0.20 ± 0.03) and D. reticulatum (0.20 ± 0.03) demonstrated strong chemoattractant properties for O. myriophilus. Meanwhile, the mucus of C. virgata (0.19 ± 0.02), T. budapestensis (0.17 ± 0.04), and H. pomatia (0.14 ± 0.02) exhibited weaker chemoattractant effects (Figure 2).

At 25 °C, the mucus from C. virgata displayed the strongest chemoattractant effect (chemotaxis index = 0.23 ± 0.04), whereas the mucus from A. vulgaris (0.15 ± 0.04), D. reticulatum (0.15 ± 0.02), T. budapestensis (0.13 ± 0.03), and H. pomatia (0.13 ± 0.03) was found to be weakly chemoattractive for O. myriophilus (Figure 2).

3.2. Comparison of Nematode Chemoattraction Towards Mollusk Mucus in a Choice Test

3.2.1. The Spanish Slug (Arion vulgaris) vs. Other Mollusk Species

The chemotaxis index for the Spanish slug (A. vulgaris) was significantly influenced by both the mollusk mucus (p < 0.01) and the interaction between the temperature and mollusk mucus (p < 0.01). However, no significant differences were observed across temporal replication (p = 0.79), spatial replication (p = 0.66), or due to temperature alone (p = 0.06) (

Table 3. Analysis of variance of the chemotaxis index of different mollusk species.

	Spanish Slug (A. vulgaris)
Factor	Df	F	p
Mollusk mucus	3	6.22	<0.01
Temperature (T)	1	3.73	0.06
Temporal replication	9	0.60	0.79
Spatial replication	2	0.41	0.66
Mollusk mucus × T	3	5.53	<0.01
Residual	61
Total (Corrected)	79
	Gray Field Slug (D. reticulatum)
Mollusk mucus	3	55.62	<0.01
Temperature (T)	1	1.21	0.28
Temporal replication	9	0.70	0.71
Spatial replication	2	0.38	0.69
Mollusk mucus × T	3	28.59	<0.01
Residual	61
Total (Corrected)	79
	Roman Snail (H. pomatia)
Mollusk mucus	3	16.62	<0.01
Temperature (T)	1	7.43	<0.01
Temporal replication	9	1.18	0.33
Spatial replication	2	0.53	0.59
Mollusk mucus × T	3	5.48	<0.01
Residual	61
Total (Corrected)	79
	Common White Snail (C. virgata)
Mollusk mucus	3	4.27	<0.01
Temperature (T)	1	58.95	<0.01
Temporal replication	9	1.42	0.20
Spatial replication	2	0.77	0.47
Mollusk mucus × T	3	1.20	0.32
Residual	61
Total (Corrected)	79
	Budapest Keeled Slug (T. budapestensis)
Mollusk mucus	3	11.92	<0.01
Temperature (T)	1	0.92	0.34
Temporal replication	9	1.02	0.44
Spatial replication	2	0.46	0.63
Mollusk mucus × T	3	8.87	<0.01
Residual	61
Total (Corrected)	79

).

At 25 °C, the motility of O. myriophilus was not significantly affected (p = 0.62) by the mucus of A. vulgaris when compared to other mollusk species. Conversely, at 20 °C, A. vulgaris mucus was less attractive to O. myriophilus than that of C. virgata and T. budapestensis (

Table 4. Effect of the mucus of different species of mollusks on the chemotaxis index of O. myriophilus at two temperatures.

Temperature (°C)	Chemotaxis Index Between Mucus Treatments
	A. v.–C. v.	A. v.–D. r.	A. v.–H. p.	A. v.–T. b.
20	−0.21 ± 0.07 Aa	−0.04 ± 0.05 Ab	0.16 ± 0.04 Bc	−0.13 ± 0.04 Aab
25	0.00 ± 0.04 Bab	−0.06 ± 0.03 Aa	0.04 ± 0.05 Abc	0.11 ± 0.05 Bc
	C. v.–A. v.	C. v.–D. r.	C. v.–H. p.	C. v.–T. b.
20	0.21 ± 0.07 Ba	0.42 ± 0.03 Bc	0.30 ± 0.05 Bab	0.34 ± 0.05 Bbc
25	0.00 ± 0.04 Aa	0.08 ± 0.04 Aab	0.07 ± 0.04 Aab	0.12 ± 0.04 Ab
	T. b.–A. v.	T. b.–C. v.	T. b.–D. r.	T. b.–H. p.
20	0.13 ± 0.04 Bd	−0.34 ± 0.05 Aa	0.00 ± 0.03 Ac	−0.12 ± 0.04 Ab
25	−0.11 ± 0.05 Aa	−0.12 ± 0.04 Ba	0.03 ± 0.05 Ab	−0.01 ± 0.05 Bab
	D. r.–A. v.	D. r.–C. v.	D. r.–H. p.	D. r.–T. b.
20	0.04 ± 0.05 Ab	−0.42 ± 0.03 Ba	−0.02 ± 0.04 Ab	0.00 ± 0.03 Ab
25	0.06 ± 0.03 Ab	−0.08 ± 0.04 Aa	−0.10 ± 0.05 Aa	−0.03 ± 0.05 Aa
	H. p.–A. v.	H. p.–C. v.	H. p.–D. r.	H. p.–T. b.
20	−0.16 ± 0.04 Ab	−0.30 ± 0.05 Aa	0.02 ± 0.04 Ac	0.12 ± 0.04 Bd
25	−0.04 ± 0.05 Ba	−0.07 ± 0.04 Ba	0.10 ± 0.05 Ab	0.01 ± 0.05 Aab

The data shown are the average values of the chemotaxis index ± S.E. The capital letters indicate statistically significant differences (p < 0.05) among the different temperatures within the same species, and the small letters indicate statistically significant differences (p < 0.05) among the different species at the same temperature. Legend: A. v.—A. vulgaris; C. v.—C. virgata; D. r.—D. reticulatum; H. p.—H. pomatia; T. b.—T. budapestensis.

). Furthermore, the mucus of A. vulgaris was found to be more attractive to O. myriophilus than that of the Roman snail (H. pomatia).

3.2.2. The Gray Field Slug (Deroceras reticulatum) vs. Other Mollusk Species

The chemotaxis index for the gray field slug (D. reticulatum) was significantly influenced by mollusk mucus (p < 0.01) and the interaction between temperature and mollusk mucus (p < 0.01). However, no significant differences were observed across temporal replication (p = 0.71), spatial replication (p = 0.69), or due to temperature alone (p = 0.28) (Table 3).

At 25 °C, O. myriophilus motility was not impacted by the presence of D. reticulatum mucus in comparison to other mollusk species (p = 0.31). In contrast, at 20 °C, D. reticulatum mucus was less attractive than C. virgata mucus (−0.42 ± 0.03). No significant differences in chemoattraction were observed among the other mollusk species tested (Table 4).

3.2.3. The Roman Snail (Helix pomatia) vs. Other Mollusk Species

The chemotaxis index for the Roman snail (H. pomatia) was significantly influenced by mollusk mucus (p < 0.01), temperature (p < 0.01), and their interaction (p < 0.01). However, no significant differences were observed across replications (Table 3).

At 25 °C, H. pomatia mucus did not significantly affect the motility of O. myriophilus when compared to other mollusk species. However, at 20 °C, H. pomatia mucus showed greater attractiveness than that of T. budapestensis (chemotaxis index = 0.12 ± 0.04) but was less attractive than the mucus of C. virgata (−0.30 ± 0.05) and A. vulgaris (−0.16 ± 0.04) (Table 4).

3.2.4. The Common White Snail (Cernuella virgata) vs. Other Mollusk Species

The chemotaxis index for the common white snail (C. virgata) showed significant variation depending on the mollusk mucus (p < 0.01) and temperature conditions (p < 0.01). However, no significant differences were detected due to replication or the interaction between temperature and treatment (Table 3).

At 25 °C, C. virgata mucus did not significantly affect the motility of O. myriophilus when compared to other mollusk species. At 20 °C, however, C. virgata mucus was more attractive than the mucus of all other tested mollusk species. The chemotaxis index values ranged from 0.21 ± 0.07 for A. vulgaris to 0.42 ± 0.03 for D. reticulatum (Table 4).

3.2.5. The Budapest Keeled Slug (Tandonia budapestensis) vs. Other Mollusk Species

The chemotaxis index for the Budapest keeled slug (T. budapestensis) was significantly influenced by mollusk mucus (p < 0.01) and the interaction between the temperature and mollusk mucus (p < 0.01). No significant differences were observed across replications or due to temperature alone (Table 3).

At 25 °C, the mucus of T. budapestensis did not significantly influence the motility of O. myriophilus, and no significant differences (p > 0.05) were detected when compared to the other mollusk species. At 20 °C, however, the mucus of T. budapestensis was less attractive to O. myriophilus than that of C. virgata (−0.34 ± 0.05) (Table 4).

4. Discussion

The effectiveness of parasitic nematodes in killing a host relies on their capacity to locate it within a limited timeframe. These nematodes identify their mollusk hosts through physical and chemical cues released by the mollusks [25]. Nematode sensory organs, specifically the amphids and inner sensilla, located on the anterior part and appearing as paired lateral structures, enable them to detect these chemical signals [38]. Despite this, research on the chemical interactions and communication between nematodes and mollusks remains limited across all mollusk parasitic nematodes [33]. Advancing our understanding of nematode responses to mollusk secretions, such as mucus, could improve their application as biological control agents.

The findings of this study revealed that the chemotactic response of O. myriophilus to mollusk mucus is significantly influenced by both temperature and the mollusk species involved. Specifically, nematode motility was consistently greater at 20 °C compared to 25 °C, suggesting that lower temperatures enhance the activity of O. myriophilus. This result aligns with previous research on other mollusk parasitic nematodes such as Phasmarhabditis species, where chemical communication and host-seeking behaviors were found to be influenced by external environmental factors, including temperature [25,31,33]. The temperature effect observed in the current study was also evident in the chemotaxis index, particularly when evaluating C. virgata mucus, which was more attractive at 20 °C compared to 25 °C. These observations suggest that temperature may influence the chemotactic behavior of O. myriophilus towards mollusk hosts, but further studies across a broader range of temperatures are needed to confirm this relationship. These preliminary findings have potential implications for optimizing biological control strategies using O. myriophilus under varying field conditions [17].

The ability of parasitic nematodes to successfully locate and infect a host is a crucial determinant of their effectiveness as biological control agents [10,35]. Nematodes use their sensory organs—such as amphids and inner sensilla—to detect physical and chemical signals from potential hosts [38]. The results presented here suggest that O. myriophilus can more effectively locate mollusk hosts at lower temperatures, which could be advantageous in temperate regions where such conditions prevail. This finding complements earlier studies on P. hermaphrodita by Rae et al. [13], who demonstrated that nematode attraction towards slug excrement and mucus was temperature-dependent, thus affecting host selection behavior. Our study adds to this body of knowledge by highlighting the critical role of environmental temperature in modulating nematode–host interactions involving O. myriophilus.

At 20 °C, C. virgata consistently exhibited the highest attractiveness to O. myriophilus, as indicated by its chemotaxis index values. This is likely due to the specific composition of the mucus produced by C. virgata, which may contain chemical cues that are highly effective at eliciting nematode responses at lower temperatures. Conversely, the mucus of T. budapestensis and H. pomatia were less attractive, particularly at 25 °C. Similar findings were reported by Andrus et al. [32], who found that Phasmarhabditis species exhibit species-specific responses to mollusk mucus based on the chemical properties of the mucus. The variations in chemotactic response seen in the current study reinforce the idea that temperature modulates not only the intensity of nematode attraction but also the relative attractiveness of different host species. Such specificity could provide an ecological advantage to nematodes like O. myriophilus in selecting optimal hosts under varying environmental conditions.

The mucus of A. vulgaris was found to be more attractive to O. myriophilus compared to H. pomatia, emphasizing the importance of differences in mucus chemical composition that influence nematode–host interactions. Similar behavioral differences were previously observed in other Phasmarhabditis species, such as P. californica and P. neopapillosa, which exhibited differential attraction to various slug mucus types [29,30]. The heightened attraction to A. vulgaris compared to H. pomatia in our study may indicate a higher concentration of specific chemoattractive compounds within A. vulgaris mucus. Understanding these interspecies differences is essential for developing biological control strategies that leverage nematode behavior to target specific pest species effectively.

The role of temperature was further emphasized in the assays involving T. budapestensis, where no significant chemotactic differences were observed at 25 °C, but lower attractiveness was noted at 20 °C compared to C. virgata. This aligns with studies on other nematode species, such as entomopathogenic nematodes (EPNs), where lower temperatures were found to enhance nematode response to volatile cues from their hosts [27,28]. Thus, it can be inferred that the chemotactic response of O. myriophilus is not only species-specific but also temperature-sensitive, affecting its ability to discriminate among different hosts.

These findings provide key insights into the behavioral ecology of O. myriophilus, emphasizing the complex interplay between environmental conditions and host selection behavior. Specifically, the nematode’s increased motility at lower temperatures suggests that cooler environments may enhance its ability to locate and infect specific mollusk hosts. This has significant implications for biological control programs, particularly in temperate climates where temperature fluctuations are common. For instance, deploying O. myriophilus under cooler conditions may increase the efficacy of pest control, especially when targeting mollusk species like C. virgata, which are shown to elicit a strong chemotactic response from the nematodes at such temperatures.

However, field validation is necessary to assess how external environmental variables and biotic factors might interact with the chemical cues emitted by mollusks. Potential limitations of our study include the controlled laboratory setting, which cannot fully replicate the complexities of field conditions [21,39]. Future research should include field trials to evaluate the practical applications of O. myriophilus, considering the soil type, nematode persistence, and potential interactions with non-target organisms. By bridging laboratory findings with real-world applications, we aim to refine the use of O. myriophilus as a sustainable alternative to chemical molluscicides.

5. Conclusions

The relationship between motility and chemotaxis is central to understanding how Oscheius myriophilus interacts with mollusk hosts and its potential as a biocontrol agent. Motility refers to a nematode’s ability to move, while chemotaxis describes its directed movement in response to chemical stimuli, such as mollusk mucus. In this study, both factors were explored to evaluate the nematode’s host-seeking efficiency.

This study found that both motility and chemotactic behavior are influenced by environmental temperature and the type of mollusk mucus present. At 20 °C, the nematodes displayed higher motility compared to 25 °C, with the mucus of Cernuella virgata consistently eliciting the highest motility and chemotactic responses across all treatments. These findings suggest a direct relationship between motility and chemotaxis: increased motility enhances the nematode’s ability to respond to chemoattractants, while chemotaxis ensures that movement is directed towards a potential host.

The focus on motility and chemotaxis in this research stems from their critical role in the nematode’s ability to locate and infect mollusk hosts. This study aimed to investigate the influence of temperature and species-specific chemical cues on these behaviors to optimize the use of O. myriophilus as a biocontrol agent. These behaviors are also key determinants of the nematode’s efficacy under field conditions. Understanding motility and chemotaxis provides insights into host-finding strategies and can help tailor biological control methods to specific environmental and pest conditions.

In summary, motility and chemotaxis are not only interrelated but also essential for the practical application of O. myriophilus in a biological control. The research findings underline the importance of these behaviors in optimizing nematode deployment strategies.