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.     

Characterization of the first molluscicidal lipopolysaccharide from Moraxella osloensis

Moraxella osloensis is a bacterium that is mutualistically associated with Phasmarhabditis hermaphrodita, a nematode that has potential for the biocontrol of mollusk pests, especially the slug Deroceras reticulatum. We discovered that purified M. osloensis lipopolysaccharide (LPS) possesses a lethal toxicity to D. reticulatum when administered by injection but no contact or oral toxicity to this slug. The toxicity of the LPS resides in the lipid A moiety. M. osloensis LPS was semiquantitated at 6 x 10(7) endotoxin units per mg. The LPS is a rough-type LPS with an estimated molecular weight of 5,300. Coinjection of galactosamine with the LPS increased the LPS's toxicity to the slug two- to four-fold. The galactosamine-induced sensitization of the slug to the LPS was reversed completely by uridine.     

Susceptibility and immune response of Deroceras reticulatum, Milax gagates and Limax pseudoflavus exposed to the slug parasitic nematode Phasmarhabditis hermaphrodita

We exposed three slug species (Deroceras reticulatum (Müller), Milax gagates (Draparnaud) and Limax pseudoflavus L.) to the parasitic nematode Phasmarhabditis hermaphrodita Schneider. P. hermaphrodita was able to cause mortality and feeding inhibition to both D. reticulatum and M. gagates but did not negatively affect L. pseudoflavus. On dissection of surviving L. pseudoflavus large numbers of P. hermaphrodita were found encapsulated in the shell of the slug. We found that by increasing shell size, the slug was able to trap invading nematodes, which could be an immune response to P. hermaphrodita invasion. This is the first report of a slug defense mechanism to inhibit P. hermaphrodita.     

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.     

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.     

A Species-specific Monoclonal Antibody System for Detecting the Remains of Field Slugs, Deroceras reticulatum (Muller) (Mollusca: Pulmonata), in Carabid Beetles (Coleoptera: Carabidae)

The field slug, Deroceras reticulatum (Muller), is probably the most damaging and widespread species of slug, causing severe economic losses in a broad range of crops in temperate zones throughout the world. Investigations into the role of predation in the population dynamics of this species required a biochemical system which was capable of identifying the remains of this slug in the crop contents of predators, and distinguishing them from those of other molluscs. A monoclonal antibody was developed (IgM isotype). This was capable of separating, in an enzyme-linked immunosorbent assay, D. reticulatum from the related D. caruanae (Pollonera) and all molluscs and other invertebrates tested, with the unexpected exceptions of New Zealand flatworms, Artioposthia triangulata (Deny) and the millipede Polymicrodon polydesmoides (Leach). Characterization of the antibody and assay demonstrated that the system could clearly detect as little as 11.6 ng of D. reticulatum protein in 200 mu l of buffer. Slug remains could be identified as such in the crops of the carabid predator Pterostichus melanarius Illiger for 38.1 h, while the antibody-antigen reaction declined to half of that measured immediately following consumption, after 12.9 h. A practical and highly sensitive system was therefore developed, using the first species-specific monoclonal antibody available for the investigation of predation on slugs.     

Infection behavior of the rhabditid nematode Phasmarhabditis hermaphrodita to the grey garden slug Deroceras reticulatum.

Infection behavior of the rhabditid nematode Phasmarhabditis hermaphrodita to the grey garden slug Deroceras reticulatum was studied. The dauer (enduring or nonaging) juveniles of P. hermaphrodita invade D. reticulatum within 8-16 hr following external exposure, with the posterior mantle region containing the shell cavity serving as the main portal of entry. The dauer juveniles can recover, multiply, and produce new dauer juveniles in the slug and slug feces homogenates, but not in the soil extract. These results demonstrate that P. hermaphrodita is a facultative parasite of the slug and can complete its life cycle under nonparasitic conditions associated with the host. Although the juvenile and adult nematodes can kill the slug if injected into the shell cavity of the host, only the dauer juvenile can serve as an infective stage in the natural environment.     

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.     

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.     

Endotoxin activity of Moraxella osloensis against the grey garden slug,Deroceras reticulatum

Moraxella osloensis is a gram-negative bacterium associated with Phasmarhabditis hermaphrodita, a slug-parasitic nematode that has prospects for biological control of mollusk pests, especially the grey garden slug, Deroceras reticulatum. This bacterium-feeding nematode acts as a vector that transports M. osloensis into the shell cavity of the slug, and the bacterium is the killing agent in the nematode-bacterium complex. We discovered that M. osloensis produces an endotoxin(s), which is tolerant to heat and protease treatments and kills the slug after injection into the shell cavity. Washed or broken cells treated with penicillin and streptomycin from 3-day M. osloensis cultures were more pathogenic than similar cells from 2-day M. osloensis cultures. However, heat and protease treatments and 2 days of storage at 22 degrees C increased the endotoxin activity of the young broken cells but not the endotoxin activity of the young washed cells treated with the antibiotics. This suggests that there may be a proteinaceous substance(s) that is structurally associated with the endotoxin(s) and masks its toxicity in the young bacterial cells. Moreover, 2 days of storage of the young washed bacterial cells at 22 degrees C enhanced their endotoxin activity if they were not treated with the antibiotics. Furthermore, purified lipopolysaccharide (LPS) from the 3-day M. osloensis cultures was toxic to slugs, with an estimated 50% lethal dose of 48 microg per slug, thus demonstrating that the LPS of M. osloensis is an endotoxin that is active against D. reticulatum. This appears to be the first report of a biological toxin that is active against mollusks.     

Characterization of the First Molluscicidal Lipopolysaccharide from Moraxella osloensis

Moraxella osloensis is a bacterium that is mutualistically associated with Phasmarhabditis hermaphrodita, a nematode that has potential for the biocontrol of mollusk pests, especially the slug Deroceras reticulatum. We discovered that purified M. osloensis lipopolysaccharide (LPS) possesses a lethal toxicity to D. reticulatum when administered by injection but no contact or oral toxicity to this slug. The toxicity of the LPS resides in the lipid A moiety. M. osloensis LPS was semiquantitated at 6 × 10⁷ endotoxin units per mg. The LPS is a rough-type LPS with an estimated molecular weight of 5,300. Coinjection of galactosamine with the LPS increased the LPS's toxicity to the slug two- to four-fold. The galactosamine-induced sensitization of the slug to the LPS was reversed completely by uridine.     

Control of slugs with contact-action molluscicides

A number of iron and aluminium compounds were compared as contact-acting poisons against the slug Deroceras reticulatum, by confining slugs on treated surfaces of glass and wet soil in laboratory tests. Simple metal salts were effective poisons when applied to a glass surface, but were rapidly inactivated when applied to wet soil. Chelation of both metals with organic ligands retarded the rate of attenuation on wet soil. In a field trial D. reticulatum and Arion spp. slugs were killed by a broadcast application of the chelate iron (III) 2,4-pentanedionate applied at 40 kg a.i. ha^-1.     

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.     

A Species of Tetrahymena Parasitic in the Renal Organ of the Slug Deroceras reticulatum

SUMMARY. A ciliate found in the renal organ of the gray garden slug, Deroceras reticulatum , is referred to Tetrahymena (Paraglaucoma) rostrata (Kahl). In the parasitic phase, the ciliate is evidently not histiophagic. It is readily cultivated in bacterized tissue infusions, and has been established in axenic culture in yeast extract.
The number of complete primary ciliary meridians in ciliates of the parasitic phase has been observed to range from 41 to 58; in ciliates from cultures, the number of meridians has been observed to range from 28 to 37. In culture, the form of T. rostrata from D. reticulatum appears to be morphologically identical with a strain (NZ-4) recovered from soil in New Zealand.     

SLUG DAMAGE AND NUMBER OF SLUGS (GASTROPODA: PULMONATA) IN WINTER WHEAT IN FIELDS WITH SOWN WILDFLOWER STRIPS

Previous observations have shown that slugs found favourableconditions for reproduction in wildflower strips and that theycaused high damage to oilseed rape adjacent to the strips. Inthe current study slug numbers and damage were estimated inwildflower strips and at different distances from the stripsinto winter wheat crops, from 1994 to 1996. Slug damage wasestimated using an index of defoliation and slug numbers andactivity were measured using wheat bran traps. Investigationsbegan when winter wheat seedlings emerged and lasted for fiveweeks. Slug damage was never severe in any of the fields studied.In most fields, slug damage was higher close to the wildflowerstrips than at greater distances from the strips. The decliningslug damage with increasing distances from the wildflowers stripswas related to the distribution pattern of juvenile Arion lusitanicus,suggesting that this species was responsible for the higherdamage near the strips. Other slug species (Deroceras reticulatum,Arion fasciatus, Deroceras laeve) were more or less evenly distributedover the field or were almost entirely confined to the wildflowerstrips. Results showed that winter wheat was not at risk fromslug feeding in spite of the establishment of wildflower stripsand that therefore the application of molluscicide along thestrips is unnecessary. (Received 23 June 1997; accepted 17 October 1997)     

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.     

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.     

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 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.     

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.