Biochemical and bioassay analysis of resistance of potato (Solanum tuberosum L.) cultivars to attack by the slug Deroceras reticulatum (Muller)

A method is described for using young field slugs Deroceras reticulatum (Muller) in a bioassay study of biochemical resistance of potato (Solanum tuberosum L.) cultivars to slugs. Tuber parts or an artificial diet were provided as food sources. Comparisons were made of feeding, survival and weight gain between the susceptible cultivar Maris Piper and the resistant cultivar Pentland Dell. Biochemical analyses were made of these two cultivars and the resistant cultivars Stormont Enterprise and Majestic. Comparisons of tuber sections and peelings as food sources indicated factors affecting growth were located in the surface layers of the tubers. Phenolics and glycoalkaloids were concentrated in the surface layers but the amounts were similar in the susceptible and resistant cultivars and the bioassays indicated that neither acting alone could explain resistance. The amounts and distribution of free amino acids also did not correlate with resistance although when supplied in the artificial diet they partly inhibited feeding. Proteinaceous inhibitors of slug gut proteolytic enzymes were present throughout the tubers but were not concentrated in the surface layers and the amounts were similar in the different cultivars thus they too did not explain the difference in susceptibility between the cultivars. Bioassays using acetone extracts (low molecular weight substances) and acetone powders (high molecular weight substances) either alone or in combination indicated that the resistant cultivar Pentland Dell contained a high molecular weight substance which together with a low molecular weight substance from either the same cultivar or the susceptible Maris Piper could confer resistance. Bioassays using protein extracts supplied in the presence or absence of chlorogenic acid indicated that this mechanism could comprise enzymic oxidation of phenolics. Assays of phenolase confirmed this since activity was highest in the outer layers of the tubers and was highest in the three resistant cultivars. Thus the chief resistance factor identified was high phenolase activity acting rapidly on phenolics when the slug first bites the tuber surface. The quantity of phenolics per se did not control the resistance. Thus while phenolics must be available, resistance is compatible with low blackening on cutting the tuber.     

Radular mechanosensory neuron in the buccal ganglia of the terrestrial slug,Incilaria fruhstorferi

A radular mechanosensory neuron, RM, was identified in the buccal ganglia of Incilaria fruhstorferi. Fine neurites ramified bilaterally in the buccal ganglia, and main neurites entered the subradular epithelium via buccal nerve 3 (n3). When the radula was distorted by bending, RM produced an afferent spike which was preceded by an axonic spike recorded at n3. The response of RM to radular distortion was observed even in the absence of Ca²⁺, which drastically suppressed chemical synaptic interactions. Therefore, RM was concluded to be a primary radular mechanoreceptor.During rhythmic buccal motor activity induced by food or electrical stimulation of the cerebrobuccal connective, RM received excitatory input during the radular retraction phase. In the isolated buccal ganglia connected to the radula via n3s, the afferent spike, which had been evoked by electrical stimulation of the subradular epithelium, was broadened with the phasic excitatory input. Since the afferent spike was also broadened by current injection into the soma, depolarization due to the phasic input may have produced the spike broadening.Spike broadening was also observed during repetitive firing evoked by current injection. The amplitude of the excitatory postsynaptic potential in a follower neuron increased depending on the spike broadening of RM.Abbreviations CBC cerebrobuccal connective - EPSP excitatory postsynaptic potential - n1,n3 buccal nerves 1 and 3 - RBMA rhythmic buccal motor activity - RM radular mechanosensory neuron - SMT supramedian radular tensor neuron     

A sialic acid-specific lectin from the slug Limax flavus

The slug, Limax flavus, contains a lectin that appears to be highly specific for sialic acid residues of glycoproteins. The carbohydrates which inhibited the hemagglutinating activity of the slug lectin and the concentration of the carbohydrate which gave a 50% inhibition are as follows: N-acetylneuraminic acid, 0.13 mm; N-glycolylneuraminic acid, 0.90 mm; d-glucosamine, 4.9 mm; d-galactosamine, 7.6 mm; N-acetyl-d-glucosamine, 23 mm; and N-acetyl-d-galactosamine, 24 mm. d-Galactose, d-glucose, d-mannose, α-methyl-d-glucoside, α-methyl-d-mannoside, l-arabinose, d-xylose, l-fucose, d-glucuronic acid, lactose, and sucrose were found to be ineffective as inhibitors of the hemagglutinating activity of the slug lectin. Hemagglutination by slug lectin was strongly inhibited by bovine submaxillary mucin and fetuin but not by sialic acid-free bovine submaxillary mucin or fetuin.     

A bioassay for screening materials influencing feeding in the field slug Deroceras reticulatum (Müller) (Mollusca: Pulmonata)

Test materials were incorporated in wheatflour pellets to measure their effect on slug feeding. The dry weights of individual pellets were measured before and after exposure for 24 h to starved slugs. The method gave consistent results, and can accommodate test materials with different physical properties. The phagostimulant effect of sucrose was confirmed. An extract of the herb tarragon (Artemisia dracunculus) was the most effective antifeedant of the materials used at the concentrations tested. The method is compared with other published techniques.     

A method to study slug predation on seedlings in the field

Dandelion is a common Asteraceae species that populates disturbed sites and gaps within swards where it becomes an important competitor of grasses. The natural control against dandelion includes seedling predation, with slugs, particularly Arion lusitanicus, being the most important in the Czech Republic. However, the study of slug seedling consumption is difficult because naturally established seedlings are not always available. Therefore, we developed a method of exposing laboratory‐grown seedlings as bait for slug predation. Dandelion seeds were sown in plastic cups containing a bottom layer of moist substrate. The emerged seedlings were thinned to 20 and displayed in an open area. During 2008–2010, the seedling baits were placed at 15 sites at 1 month intervals throughout the dandelion vegetative season, in parallel with plasticine baits that monitored slug feeding activity. For each 1 month interval, seedling survival was observed for a period of 8 days, and the estimated time to death was calculated; the percentage of surviving seedlings was then recorded. This method of seedling presentation demonstrated that local and temporal variation in seedling survival is correlated with slug feeding activity. The advantage of this technique is that the seedlings in baits may be presented at any place and time, as required by the experimental design; however, we found that the estimated time to seedling death was shorter for the exposed baits than for the naturally established seedlings. The method is suitable for the study of plant species other than dandelion, and also for aims other than the study of spatiotemporal trends in seedling consumption by slugs.     

Transgenic Arabidopsis leaf tissue expressing a modified oryzacystatin shows resistance to the field slug Deroceras reticulatum (Müller)

Transgenic Arabidopsis thaliana has been developed which expresses the oryzacystatin mutant OCI86, which is an inhibitor of the major proteinase present in the digestive gland of the slug, Deroceras reticulatum. When fed on leaf tissue from plants expressing this inhibitor the growth of juvenile slugs was significantly reduced by 31% compared with those feeding on control leaf tissue. Furthermore, while surviving slugs did not individually consume less when feeding on leaf tissue expressing OCI86, the total amount of leaf tissue eaten was 50% less, due to reduced survival of slugs. The synthetic cysteine proteinase inhibitors E64 and leupeptin also significantly reduced slug weight gain (by at least 40%) and digestive gland cysteine proteinase activity when administered in an artificial diet, indicating that their antimetabolic effects are due to direct inhibition of gut proteolytic activity. These results suggest that transgenic crop plants expressing phytocystatins could be used to suppress the growth rates of slug populations in the field.     

Effects of plant extracts on the feeding behaviour of the slug Arion lusitanicus

Methanol extracts of the plants Geranium robertianum, Lepidium sativum, Origanum vulgare, Salvia officinalis, Salvia pratensis, Saponaha officinalis, Thymus vulgaris, Trifolium repens and Valerianella locusta were produced and applied to cotyledons of 6–8 days old rape seedlings to investigate whether they can influence the feeding of the slug Arion lusitanicus on young rape. All plant extracts appeared to reduce slug feeding on rape to some extent, but only the extracts of Saponaria officinalis and V. locusta significantly deterred A. lusitanicus from feeding. The small effects of some extracts may be due to a poor extraction efficiency or an insufficient feeding inhibition capacity of these extracts.     

Use of lichen secondary metabolites as antifeedants to protect higher plants from damage caused by slug feeding

Extracts of 15 species of lichen were tested for antifeedant properties against the field slug Deroceras reticulation (Müller) (Mollusca: Pulmonata). All but three showed some activity and two of these were from endolithic species. The most effective extract was from Letharia vulpina. The major active compound from this species, vulpinic acid, was tested for dose response, applied as a foliar spray to turnip plants and as a dressing to wheat seeds in laboratory experiments. It provided effective protection against slug feeding under these conditions.     

Glutamate induces Cl− and K+ currents in the olfactory interneurons of a terrestrial slug

Glutamate-induced responses in the procerebral neurons of the terrestrial slug Limax marginatus were examined using the nystatin-perforated patch recording technique applied in the voltage-clamp mode and local application of drugs. The procerebrum contains two types of interneurons with different spontaneous activities, bursting and nonbursting neurons. In the bursting neurons, a puff of glutamate evoked a rapidly desensitizing current followed by a smaller sustained current. The reversal potential of the early component showed that the current was mediated by Cl⁻ ions, while the late component was presumed to be mediated by K⁺ ions. In the nonbursting neurons, glutamate evoked a sustained current with a strong outward rectification, and the current was mediated by K⁺ ions. Ibotenate selectively evoked the rapidly desensitizing response in the bursting neurons, whereas quisqualate evoked a non-desensitizing K⁺ current both in the bursting and nonbursting neurons. The glutamate-induced K⁺ current had similar characteristics with the spontaneous synaptic activities in the procerebrum neurons, suggesting the possibility that glutamate receptors are involved in the spontaneous oscillatory activity. Accepted: 10 February 1999     

Plant characters related to slug feeding: a re‐analysis of feeding studies using trait data from botanical databases and literature

Slugs feed on some plant species but not on others for reasons that are not yet fully understood. We re‐examined the two largest studies on the preference of Arion lusitanicus for different plant species. Briner & Frank (1998) measured the consumption index of slugs (CI_B) based on the dry leaf mass eaten per day per gram of slug mass. Kozlowski & Kozlowska (2009) also measured a consumption index of slugs (CI_K) but on the basis of the area eaten per day per gram of slug mass. The two indices showed a moderate positive rank correlation. Briner & Frank (1998) found that slugs preferred annual to perennial plants and crops rather than wild species. On the other hand, Kozlowski & Kozlowska (2009) found no statistically significant difference in slug preference for annuals, biennials or perennials. Using botanical databases we selected plant traits related to slug herbivory and analysed 141 species. We first analysed the two data sets separately and found that CI_B was higher for seedlings with a higher relative growth rate although this result was only marginally statistically significant. In addition, we found that annuals were consumed more than perennials and that cultivated plants were consumed more than wild plants. This is in agreement with the conclusions of Briner & Frank (1998). For CI_K slugs consumed less from plants with high dry matter content (DMC). The use of classification and regression tree models solves the problem of analysing datasets with missing values. Regarding CI_B, the tree models indicated that life cycle was the major branch. Within perennials, cultivated/wild origin of plants was the most important predictor of slug feeding. Within wild perennials slugs ate more from plants with low specific leaf area (SLA), i.e. relatively thick leaves. Within annuals we found the unexpected result that slugs ate more from plant species with either alkaloids or glucosinolates than from species without one of these compounds. For CI_K, the results of tree models showed that slugs preferred shade‐intolerant plants to shade‐tolerant plant species. Within the shade‐tolerant group slugs fed more on leaves with low DMC. We only partially understand which factors make plants acceptable to slugs. We discuss this issue along with the pros and cons of the two methods used to calculate CI.