Effects of soil incorporation on the efficacy of the rhabditid nematode, Phasmarhabditis hermaphrodita, as a biological control agent for slugs

The effects of soil cultivation immediately after application of the rhabditid nematode, Phasmarhabditis hermaphrodita , to the soil surface were investigated in two field experiments. The first experiment was done in mini-plots separated by barriers, with an artificially introduced population of slugs ( Deroceras reticulatum ). Nematodes were applied as a drench at a rate of 3 times 10⁹ ha^-1 in one of two application volumes and then left undisturbed or incorporated into the soil by cultivation to 2 cm or 10 cm depth. Moist soil conditions were maintained by irrigation throughout the experiment. Nematode application significantly reduced slug damage to Chinese cabbage seedlings throughout the 7 wk duration of the experiment and the population of D. reticulatum in soil 7 wk after treatment. However, soil cultivation had no effect and did not interact with the effect of nematodes. In the second experiment, in a crop of winter wheat, nematodes were applied to soil by hand-lance at a rate of 3 times 10⁹ ha^-1 and left undisturbed on the soil surface or incorporated by spring-tine cultivation to a depth of 2, 5 or 10 cm. In this experiment, nematodes were applied to dry soil. Cultivation alone had no effect. Nematode application reduced slug damage to wheat plants in plots where nematodes were incorporated into the soil, but not where they were left on the surface. There was no detectable impact of nematodes on slug populations in the wheat experiment.     

Biocontrol of Slugs in Protected Lettuce Using the Rhabditid Nematode Phasmarhabditis hermaphrodita

In two experiments, the rhabditid nematode Phasmarhabditis hermaphrodita, a parasite of slugs, was cultured in vitro and applied as a drench to soil at four dose rates (3 108, 1 109, 3 109 and 1 1010 ha-1) 1 or 4 days before planting lettuce seedlings in a polythene tunnel. The effects of the four nematode doses on slug damage during the first 3 weeks after planting and on the numbers of slugs found within and below lettuce plants at harvest were measured. Results were then compared with untreated plots and with plots where methiocarb pellets were applied at the recommended field rate. In the first experiment, methiocarb pellets significantly reduced the percentage of plants damaged by slugs, but the nematode did not. In the second experiment, methiocarb pellets and the second highest dose of nematodes significantly reduced the percentage of plants damaged by slugs. The different effect of the nematode in the two experiments may have resulted from differences in the timing of nematode application and/or differences in the pattern of slug damage between experiments. At the end of the first experiment, the highest two doses of nematodes and methiocarb pellets had significantly reduced the number of slugs found within lettuce plants at harvest and on the soil surface below the plants. At the end of the second experiment, analysis of variance showed no significant effects of any treatment on slug numbers or biomass, but regression analysis showed significant negative relationships between nematode dose and total slug numbers, numbers of Arion ater agg. and biomass of Deroceras reticulatum. In both experiments, increasing nematode dose significantly reduced the numbers of slugs found contaminating the harvested lettuce. At the end of the second experiment, the mean weight of individuals of A. ater agg. increased with rising nematode dose.     

The influence of nutritional status on the feeding behaviour of the field slug, Deroceras reticulatum (Müller)

The field slug, Deroceras reticulatum, a common pest of agricultural and horticultural crops, is a generalist herbivore with distinct preferences for particular food items. However, these preferences are not fixed, but are influenced by the recent dietary history of the slugs. In particular, slugs tend to select novel food items ('neophilia'). We investigated the basis of such influences, using artificial diets in which protein and carbohydrate composition could be controlled and non-nutritional ('secondary') chemicals added as flavours. The slugs showed no general inclination for neophilia based on taste alone. There was a possible learned association between protein content and taste, but this was weakly expressed. However, the slugs selected food items, when given a choice, containing nutrients that were deficient in earlier diets, even though all the food items contained the same familiar secondary taste chemicals. Injections of missing nutrients into the haemocoel of slugs inhibited such changes in food preference, indicating that slugs' feeding preferences are influenced directly by their internal nutritional status. We suggest that neophilia in D. reticulatum is a physiological response to a nutritional imbalance arising from a suboptimal diet. Copyright 2000 The Association for the Study of Animal Behaviour.     

Spatial sampling to detect slug abundance in an arable field

Overuse of molluscicides by farmers in arable systems can lead to environmental and product contamination. Here we assess a simple and inexpensive surface trapping method for monitoring populations of slugs (Deroceras reticulatum and Arion intermedius). This method was biased against small slugs, and against A. intermedius, when compared to direct soil sampling. Regression was used to model the relationship between the results of surface trapping and soil sampling methods. Spatial Analysis by Distance IndicEs (SADIE) algorithms were used to describe the spatial relationships between the two sets of samples. Using both traditional statistical methods and spatial statistics, the spatial information collected from surface traps was sufficient to identify patches and gaps in slug numbers and possibly to allow the spot application of slug control, and thus provide land managers who experience slug damage with a way of reducing molluscicides use, whilst maintaining slug control. Further improvements and applications of the model are discussed.     

Pathogenicity of Moraxella osloensis, a bacterium associated with the nematode Phasmarhabditis hermaphrodita, to the slug Deroceras reticulatum.

Moraxella osloensis, a gram-negative bacterium, is associated with Phasmarhabditis hermaphrodita, a nematode parasite of slugs. This bacterium-feeding nematode has potential for the biological control of slugs, especially the grey garden slug, Deroceras reticulatum. Infective juveniles of P. hermaphrodita invade the shell cavity of the slug, develop into self-fertilizing hermaphrodites, and produce progeny, resulting in host death. However, the role of the associated bacterium in the pathogenicity of the nematode to the slug is unknown. We discovered that M. osloensis alone is pathogenic to D. reticulatum after injection into the shell cavity or hemocoel of the slug. The bacteria from 60-h cultures were more pathogenic than the bacteria from 40-h cultures, as indicated by the higher and more rapid mortality of the slugs injected with the former. Coinjection of penicillin and streptomycin with the 60-h bacterial culture reduced its pathogenicity to the slug. Further work suggested that the reduction and loss of pathogenicity of the aged infective juveniles of P. hermaphrodita to D. reticulatum result from the loss of M. osloensis from the aged nematodes. Also, axenic J1/J2 nematodes were nonpathogenic after injection into the shell cavity. Therefore, we conclude that the bacterium is the sole killing agent of D. reticulatum in the nematode-bacterium complex and that P. hermaphrodita acts only as a vector to transport the bacterium into the shell cavity of the slug. The identification of the toxic metabolites produced by M. osloensis is being pursued.     

Effects of seed-bed conditions on slug numbers and damage to winter wheat in a clay soil

In a field experiment three levels of consolidation were combined with three seed-bed tilths, to produce nine different types of seed-bed. The soil properties of each combination of tilth and consolidation were measured and effects on the numbers and biomass of slugs in the top 10 cm of soil and on damage to wheat seeds and seedlings were assessed. Eight species of slug were present, with three species predominating (Deroceras reticulatum, Arion distinctus and Arion subfuscus). Between 3% and 33% of seeds and seedlings were killed by slugs (recorded at Growth Stage 12). Contrary to expectation, most damage occurred on consolidated seed-beds of fine or medium tilth, least on loose seed-beds of the same texture; intermediate damage occurred on cloddy seed-beds, where consolidation had little effect. The level of damage occurring on different types of seed-bed was directly related to the biomass of slugs in the top 10 crn of soil, and was inversely related to depth of sowing and the percentage of fine soil in the seed-bed. These three factors together accounted for 94–97% of the variance in slug damage. Consolidation was associated with increased slug damage probably because of its effects on these three factors: slug biomass was greater and seed was at shallower depth in consolidated than loose seed-beds and consolidation, whether before or after drilling, failed to break down clods of soil into finer particles. This is the first experimental evidence in the field of the effects of seedbed conditions on slug numbers and damage to winter wheat and represents a significant step towards forecasting and avoiding slug damage in this crop.     

The effects of crop development upon slug distribution and control by Abax parallelepipedus (Coleoptera: Carabidae)

The polyphagous carabid beetle Abax parallelepipedus has been shown to be capable of controlling slugs within a lettuce crop in previous studies. This report describes experiments undertaken in plots within a polythene tunnel. Field slugs, Deroceras reticulatum, were introduced to plots containing either large or small lettuce plants, at 30 slugs per plot. The plots were further subdivided into those with or without six beetle predators. Slug numbers were assessed after two weeks, and were found to be affected by plant size, both with and without the presence of predators. These two factors had an additive effect, with both small plant size and the presence of beetles causing significant reductions in slug numbers. Plots with beetles contained either males or females, and females were shown to be significantly better at reducing slug numbers than males, particularly within plots of small plants. The four principal treatments (combinations of two plant sizes and the presence/absence of beetles) generated four distinct patterns of slug distribution within the plots. The potential of the residual slug populations to inflict further damage was measured in a subsequent crop of seedling lettuces. It appeared that the beetles were generally incapable of capturing slugs within large lettuce plants, but were effective predators at soil level. Possible reasons for the survival of fewer slugs in plots with small plants, both with and without the presence of predators, are discussed.     

Use of an individual-based model to forecast the effect of climate change on the dynamics, abundance and geographical range of the pest slug Deroceras reticulatum in the UK

Slugs are serious agricultural pests and their activity is strongly driven by ambient temperature and soil moisture. The strength of this relationship has been shown through the development of a deterministic model, based upon temperature and soil moisture conditions alone, which accurately describes the population dynamics and abundance of Deroceras reticulatum . Because of this strong climatic dependence, slug abundance and dynamics are likely to be affected by climate change. We used a validated individual-based model (IbM) of D. reticulatum , to assess the effects of climate change on the abundance of this species in the UK. Climatic scenarios were based on the UKCIP02 predictions and constructed using the LARS-WG stochastic weather generator. The IbM of slugs predicted population dynamics at three time slices (2020s, 2050s and 2080s), and two scenarios of greenhouse gas emissions. The maximum generation number, the number of population peaks, the number of slug-days in each season, the percentage of years when the population passes over a threshold for damage and the percentage of years in which populations go extinct were investigated. Currently, the south-west of the UK has the best conditions for D. reticulatum to thrive, with the north-east of Scotland having the most adverse. By 2080 under both low- and high-emissions scenarios, the north and west of Scotland will have the most favourable conditions for the survival of this species and the east of the UK and Scotland will have the harshest. By 2080 the climate in the north-west of Scotland will become more like the current climate in south-east England, which explains the shift in the pattern of abundance. The north-west of Scotland will have increased slug damage and south-west England and west-Wales will have decreased slug damage with some changes becoming evident by 2020.     

The rhabditid nematode Phasmarhabditis hermaphrodita as a potential biological control agent for slugs

A nematode, Phasmarhabditis hermaphrodita, known to be associated with slugs but not previously thought to be parasitic, was shown to be a parasite capable of killing the pest slug Deroceras reticulatum. The parasite infects slugs in the area beneath the mantle surrounding the shell, causing a disease with characteristic symptoms, particularly swelling of the mantle. Infection leads to death of the slug, usually between seven and 21 days afterwards. The nematode then spreads and multiplies in the cadaver. In an experiment where individual D. reticulatum were exposed to different numbers of P. hermaphrodita, a significant positive relationship was found between nematode dose and slug mortality. In two experiments on host range, the nematode was found to infect and kill all pest slug species tested: Deroceras caruanae, Arion distinctus, Arion silvaticus, Arion intermedius, Arion ater, Tandonia sowerbyi and T. budapestensis, in addition to D. reticulatum.     

Experimental Infection of the Gray Garden Slug, Deroceras reticulatum (Müller), By the Holotrichous Ciliate Tetrahymena pyriformis (Ehrenberg)

SUMMARY. To determine whether Tetrahymena pyriformis could infect Deroceras reticulatum and become morphologically modified in the same way as the parasitic phase of T. limacis , 50 slugs were exposed for 10 days to an amicronucleate clone of T. Pyriformis and 50 were exposed for the same period of time to a micronucleate clone. The slugs were then transferred to clean containers for another 10 days, to allow ciliates which had entered the slugs either to persist and multiply or, in the case of transitory infections, to disappear. An additional 50 slugs were kept in a control group for 20 days. All of the slugs had been collected at a single locality in which natural infections by T. limacis had not been noted, and were maintained in separate one-half pint milk bottles throughout the experimental period.
Of the slugs exposed to the amicronucleate clone, 40 survived to the end of the experimental period, and 15 were infected. Of those exposed to the micronucleate clone, 42 survived, and 5 were infected. Forty-three of the slugs in the control group survived, and none were infected by ciliates. In some of the slugs infected by T. pyriformis , hundreds of ciliates were noted. The infections were apparently limited to the digestive gland, intestinal tract, and fluid around the viscera. Ciliates recovered from slugs were in general smaller than those growing in thriving cultures, but morphologically were typical T. pyriformis. None were observed to have a proportionately smaller cytostome or apiculate anterior end characteristic of the parasitic phase of T. limacis.     

Effect of predator size and temperature on the predation of Deroceras reticulatum (Muller) (Mollusca) by carabid beetles

Twenty-one carabid species were used in a laboratory study to determine the effect of beetle size and temperature on the predation of one day old Deroceras reticulatum (Muller) slugs. The beetles were caught in arable land at the study site. None of the five small-sized beetle species killed slugs, whereas five of the seven medium-sized beetle species and eight of the nine large-sized beetle species killed slugs. Four of the most abundant beetle species at the study site were used to assess the effect of temperature on the predation of 1-day-old slugs. There were significant differences in the proportion of beetles predating slugs at the different temperatures. Regression analysis showed that there was a significant increase in the proportion of Pterostichus madidus (Fabricius) and Harpalus rufipes (Degeer) beetles predating slugs as temperature increased. Nebria brevicollis (Fabricius) inflicted its highest level of slug mortality at 8°C and its larvae were the only predators to significantly increase slug mortality at 4°C, the lowest experimental temperature. Harpalus aeneus (Fabricius) inflicted significant levels of slug mortality at 20°C, the highest experimental temperature. It appeared that the predatory activity of these beetle species at the five experimental temperatures reflected their annual activity periods in the field.     

Complementary video and acoustic recordings of foraging by two pest species of slugs on non-toxic and molluscicidal baits

The effects of adding the molluscicides methiocarb and metaldehyde to pelleted foods on the encounter, acceptance, feeding and post-meal stages of the foraging sequence of the slugs Deroceras reticulatum and Arion distinctus have been studied using two complementary laboratory techniques - video and acoustic recordings. Whereas non-feeding slugs encountered wheat grains at random, slugs which fed on wheat grains or pellets encountered them more frequently because of changes in locomotor pattern and olfactory attraction. Slugs would almost always feed on the first pellet they found, regardless of the presence or absence of molluscicide: thereafter they fed on only one pellet in every four encountered. Far fewer slugs accepted wheat grains. Slugs ate much less from pellets containing molluscicide, and meals were more irregular. The relative amounts of non-toxic pellets of various types which were eaten gave no indication of the relative amounts eaten when molluscicide was added. Meal length did not correlate well with meal size on different pellet types because softer pellets were eaten faster. Most Deroceras fed several times on non-toxic pellets or pellets containing methiocarb, although subsequent meals were shorter than the first meal. Most returned to shelters by dawn. In contrast, metaldehydefed slugs were rapidly immobilised; they seldom fed again, moved little, and few regained shelter. Arion were also inhibited after methiocarb meals but Deroceras were not. Arion distinctus moved less, ate less during a meal, and had fewer meals on nontoxic baits than Deroceras reticulatum.     

Emergence of third-stage larvae of Umingmakstrongylus pallikuukensis from three gastropod intermediate host species

We investigated the emergence of third-stage larvae (L3) of Umingmakstrongylus pallikuukensis from the slugs Deroceras laeve, Deroceras reticulatum, and the snail Catinella sp. in the laboratory and from D. laeve on the tundra. Third-stage larvae emerged from 8 of 8 D. laeve and 8 of 8 D. reticulatum housed at 20 C in darkness and from 9 of 10 D. laeve and 5 of 5 Catinella sp. housed at 21 C with 10-12 hr of light/day. Larvae emerged from D. laeve and D. reticulatum over a wide range of infection intensities (2-179 and 20-65, respectively), and the patterns of emergence were independent of intensity. The majority of the L3 emerged from most of the Deroceras spp. by 58 or 60 days postinfection (PI). Lower rates of emergence were observed from Catinella sp. Larvae emerged from D. laeve on the tundra by 10 wk PI and were recovered from the vegetation in some experimental enclosures the following year. Third-stage larvae survived in tap and distilled water at 0-4 C for 13 mo. Emergence of L3 of U. pallikuukensis from the intermediate host may increase the temporal and spatial availability of L3 and enhance their survival and transmission.     

Daily activity rhythm and use of shelter in the slugs Deroceras reticulation and Arion distinctus under laboratory conditions

Daily activity of slugs, Deroceras reticulatum and Arion distinctus, was studied in the laboratory in relation to the presence of shelters so as to define optimal conditions for using traps to forecast crop damage. Under constant temperature and humidity, activity of slugs began with the lights off. Its arrest occurred after a period of activity with the lights on. Slugs rested most frequently under a shelter on bare earth and rarely used the same shelter for more than two consecutive days. When food was placed under the shelter, the number of slugs found under the shelter was more constant throughout the 24 h period, but lower during diurnal rest. The presence of 4% methiocarb pellets under the shelter led to an increased occupation by slugs, due to the poisoning of some of them. Acts of aggression were more numerous in A. distinctus than in D. reticulatum. In D. reticulatum the smallest slug was the least aggressive and suffered more attacks from the other slugs. In A. distinctus the numbers of attacks suffered or provoked varied little with the size of the slug, but the smallest slug showed less interactions with other slugs.     

The importance of temperature and moisture to the egg-laying behaviour of a pest slug, Deroceras reticulatum

Oviposition behaviour is important when modelling the population dynamics of many invertebrates. The numbers of eggs laid are frequently used to describe fecundity, but this measure may differ significantly from realised fecundity. Oviposition has been shown to be important when describing the dynamics of slug populations, which are important agricultural pests. The numbers of eggs laid by Deroceras reticulatum and their viability were measured across a range of 16 temperature (4, 10, 15 and 23°C) by moisture (33%, 42%, 53% and 58% by dry soil weight) experimental combinations. A fitted quadratic response surface model was used to estimate how D. reticulatum adjusted its egg laying to the surrounding temperature and moisture conditions, with most eggs being laid at a combination of 53% soil moisture and 18°C. The number and proportion of viable eggs also covaried with temperature and moisture, suggesting that D. reticulatum may alter their investment in reproduction to maximise their fitness. We have shown that the number of viable eggs differs from the total number of eggs laid by D. reticulatum . Changes in egg viability with temperature and moisture may also be seen in other species and should be considered when modelling populations of egg-laying invertebrates.     

The Influence of Mucus Production by the Slug,Deroceras reticulatum,on Predation by Pterostichus madidus and Nebria brevicollis (Coleoptera: Carabidae)

Slugs are important pests in many agricultural crops and potential biological control agents are being studied as an alternative to molluscicides. Carabid beetles may be able to reduce slug populations, but the defence mucus of slugs may deter some predator attacks. Interactions between the carabids Pterostichus madidus (Fabricius) and Nebria brevicollis (Fabricius) with healthy and 'stressed' (unable to produce defence mucus) Deroceras reticulatum (Müller) were investigated in laboratory conditions. Both beetle species consumed significantly more stressed slugs than controls. Defence mucus production by control slugs hampered beetle attacks. These generalist beetle species did not direct their attacks at vulnerable parts of the prey as equal numbers of contacts were made on the slug head, mantle and tail. Blowfly ( Calliphora sp.) larvae are readily consumed by P. madidus and N. brevicollis . Calliphora larvae coated in slug defence mucus were less acceptable to both beetle species compared with control larvae. Results indicate that these generalist beetle species are unable to overcome the defence mucus production of healthy slugs. Slugs sub-lethally poisoned by molluscicides may be a more suitable prey item due to a reduction in defence mucus production.     

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.     

Predation and prey size choice by the carabid beetle Pterostichus melanarius (Coleoptera: Carabidae): the dangers of extrapolating from laboratory to field

The impact of predation by the generalist carabid beetle Pterostichus melanarius (Illiger) on populations of the field slug Deroceras reticulatum (Müller), and the effects of prey size on the predator-prey interaction, were measured under semi-field conditions. It was hypothesized that environmental heterogeneity would lead to very different patterns of comparative mortality than might be deduced from size choice experiments conducted in the laboratory. Results from outdoor mini-plots, emulating conditions in a field of wheat, demonstrated that P. melanarius significantly reduced numbers of slugs from all size classes, with no apparent preferences. This was in marked contrast to results from earlier laboratory studies, where this beetle fed preferentially on the smallest slugs. The slugs in the mini-plots ranged in size from 2-100 mg and the numbers in the mini-plot reflected the size frequency distribution in the field. Beetles in mini-plots containing high densities of slugs increased significantly in weight, in contrast to beetles in mini-plots with low slug density or no added slugs, which did not. Enzyme-linked immunosorbent assays (ELISA), using anti-slug monoclonal antibodies, showed that where there was a higher density of slugs there was more slug protein in the guts of the beetles. It was concluded that environmental heterogeneity probably provided a greater number and diversity of refugia for smaller than for larger slugs, counteracting laboratory-measured size preferences measured in arenas without refugia. These results have implications for a range of ecological studies involving inter- and intra-specific prey size choice, and emphasize the dangers of extrapolating from the laboratory to the field.     

Molecular analysis of predation by carabid beetles (Carabidae) on the invasive Iberian slug Arion lusitanicus

The invasive Iberian slug, Arion lusitanicus, is spreading through Europe and poses a major threat to horticulture and agriculture. Natural enemies, capable of killing A. lusitanicus, may be important to our understanding of its population dynamics in recently invaded regions. We used polymerase chain reaction (PCR) to study predation on A. lusitanicus by carabid beetles in the field. A first multiplex PCR was developed, incorporating species-specific primers, and optimised in order to amplify parts of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of large Arion slugs, including A. lusitanicus from the gut contents of the predators. A second multiplex PCR, targeting 12S rRNA mtDNA, detected predation on smaller Arion species and the field slug Deroceras reticulatum. Feeding trials were conducted to measure the effects of digestion time on amplicon detectability. The median detection times (the time at which 50% of samples tested positive) for A. lusitanicus and D. reticulatum DNA in the foreguts of Carabus nemoralis were 22 h and 20 h, respectively. Beetle activity-densities were monitored using pitfall traps, and slug densities were estimated using quadrats. Predation rates on slugs in the field by C. nemoralis in spring ranged from 16-39% (beetles positive for slug DNA) and were density dependent, with numbers of beetles testing positive being positively correlated with densities of the respective slug species. Carabus nemoralis was shown to be a potentially important predator of the alien A. lusitanicus in spring and may contribute to conservation biological control.