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.     

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°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°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 μg 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.     

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.     

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.     

Size-specific susceptibility of the pest slugs Deroceras reticulatum and Arion lusitanicus to the nematode biocontrol agent Phasmarhabditis hermaphrodita

The nematode Phasmarhabditis hermaphrodita is a commercially available biocontrol agent against slugs. This product is especially interesting for use in organic farming, where products containing metaldehyde or carbamates cannot be used for controlling pest slugs. We investigated the potential of P. hermaphrodita for the control of the pest slugs Deroceras reticulatum and Arion lusitanicus. These two species are the most harmful slug pests in Switzerland. At different times of the year, we collected slug specimens of different weight and assessed their susceptibility to P. hermaphrodita in the laboratory. Batches of five slugs were subjected to five different doses of nematodes plus an untreated control and replicated three times. During six weeks, feeding and survival of the slugs were recorded. D. reticulatum was strongly affected by increasing nematode doses, irrespective of the slugs' body weight. In small specimens of A. lusitanicus, feeding and survival were strongly affected by the nematodes, while larger specimens remained almost unaffected. Because A. lusitanicus has an asynchronous development in Switzerland, it seems difficult to control the entire population with a single nematode application. To what extent nematodes will be used in practice for slug control depends on their effectivity against the pest slugs of major importance, on the longevity of the molluscicidal effect and on the price of nematodes.     

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.     

Targeting Biocontrol with the Slug-Parasitic Nematode Phasmarhabditis hermaphrodita in Slug Feeding Areas: a Model Study

The nematode Phasmarhabditis hermaphrodita was applied to soil in an outdoor miniplot experiment to protect Chinese cabbage seedlings from damage by the field slug Deroceras reticulatum. The aim was to investigate the possibility of reducing the numbers of nematodes applied by only partially spraying soil in the area where slug control was needed. Nematodes sprayed as overall applications were compared with band applications along plant rows and spot applications around individual plants, in plots with nine or 18 plants. Band and spot applications were applied at two rates, designated the full rate (same number of nematodes per plot as in the overall application) and the area rate (same number of nematodes per unit area comprising 43% (band) and 18% (spot) of the overall application). In plots with 18 plants, where spot-treated plant alternated with untreated plants, no significant difference in damage was found between spot-treated plants and untreated plants. This indicates that slugs were not repelled from nematode-treated areas and that any effects in reducing slug damage were not due to repellency. All nematode treatments resulted in a significant reduction in the mean level of slug damage to seedlings from six or more days after treatment. However, there were significant interactions between nematode treatment, the number of plants per plot, the position of plants within plots (edge or middle) and time after treatment. The effect of time after treatment was modelled. The log time to 50% reduction in slug damage (t 50 ) was related to the area treated and the dose applied. In plots with band or spot treatments at the full dose, there was a relatively small increase in t 50 with declining area treated. In plots treated with band or spot treatments at the area dose, t 50 increased consistently with declining relative area treated. The final level of damage, expressed as a percentage of damage on untreated plots (P), was influenced by both the dose and area treated. Final damage was greatest on spoti treated plots where half the plants were untreated. We conclude that partial treatment of soil around all plants to be protected from slug damage is a potentially valuable method of reducing the overall nematode dose required for control of slug damage, provided that some damage can be tolerated.     

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.     

Field Test Using the Nematode Phasmarhabditis hermaphrodita for Biocontrol of Slugs in Spain

An experiment was carried out between May and July 1999 in Galicia (North-West Spain) to test the capacity of the nematode Phasmarhabditis hermaphrodita to protect field grown lettuces from slug damage in our field conditions. The experiment compared a single dose of nematodes (3 ×109 ha -1) with mini-pellets containing 5% metaldehyde, applied at the recommended field rate (3 g pellets m -2), and untreated plots. Slug damage for each lettuce head was estimated on six dates during the first 4 weeks after planting. At harvest, each lettuce head was weighed, scored as marketable or not by weight and external aspect, and inspected for slugs. Metaldehyde significantly reduced slug damage to lettuce plants from the first day after planting to the third week. Nematodes significantly reduced slug damage from the second to the third week. At harvest, 6 weeks after planting, the mean weight of the lettuce heads and the number of marketable heads in the nematode plots were as good as in the metaldehyde plots, and both treatments were significantly better than the untreated plots. The number of slugs within the harvested plants was significantly reduced only with the metaldehyde treatment.     

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.     

Microsporidium novacastriensis n. sp., a Microsporidian Parasite of the Grey Field Slug,Deroceras reticulatum1,2

A new species of microsporidium (phylum Microspora), Microsporidium novacastriensis n. sp., from the grey field slug, Deroceras reticulatum, is described on the basis of light and electron microscope studies. Meronts are spherical at first, then become irregular as nuclear number increases. Sporonts are tubular or ribbon-like and divide unevenly to produce sporoblasts and then spores of varying lengths. Sporogonial stages are enclosed in a vesicle by a subpersistent membrane of uncertain origin. Fresh spores measure 3.5 by 2.08 μm and are produced in clusters of 12 to 120. The parasite infects only the intestinal epithelium of the slug. The new species is compared to microsporidia of other gastropod molluscs and to other microsporidia of similar developmental pattern and morphology.     

Low Temperatures Suppress Growth of the Ciliate Parasite, Tetrahymena rostrata , and Pathogenicity to Field Slugs, Deroceras reticulatum

The slug parasitic ciliate protozoan, Tetrahymena rostrata , was isolated from the slug Deroceras reticulatum and used in bioassays against D. reticulatum at 10 and 17oC and in in vitro growth experiments at a range of temperatures. The parasite reproduced, and significantly reduced the life span of juvenile D. reticulatum at 17oC, but not at 10oC.     

Effect of Gamma Irradiation on the Preference Behavior and Lipid Metabolism in Grey Garden Slug Deroceras reticulatum Mull.

We studied the preference behavior of slugs Deroceras reticulatum in experimental light, thermal, and salt gradients. The zone of each gradients preferred by the majority of individuals had the lowest contents of total lipids and total fatty acids in the tissues as compared to animals in any other zones. Hard gamma irradiation of slugs D. reticulatum proved to induce qualitative changes in salt preference, to decrease photoresponse in light gradients, and to increase lipid content in tissues.     

Mass cultivation and storage of the rhabditid nematode Phasmarhabditis hermaphrodita,a biocontrol agent for slugs

The rhabditid nematode Phasmarhabditis hermaphrodita (a parasite capable of killing pest slugs) was grown in vitro, in association with a mixed bacterial flora on foam chips impregnated with a kidney‐based nutrient medium in aerated bags, to provide sufficient numbers for laboratory and field experiments. The feasibility of producing nematodes in liquid culture was investigated using 250 ml flasks. Baffled flasks containing 25 or 50 ml or liquid were found to be better than baffled flasks containing 100 ml or unbaffled flasks. Inoculum densities ranging from 50 to 330 ml^‐1 did not affect final yield. Dauer larvae in aerated water died rapidly at temperatures of 26–35°C. Survival was progressively better at 22°C and 15°C, and best at 5°C or 10°C.     

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.     

Overall Versus Band Application of the Nematode Phasmarhabditis hermaphrodita With and Without Incorporation into Soil, for Biological Control of Slugs in Winter Wheat

In two concurrent field experiments, the effects of three types of soil cultivation and two patterns of nematode application were studied in order to investigate their effects on damage to winter wheat by slugs (assessed at Zadoks Growth Stage 12). In experiment 1, infective juveniles (IJs) of the nematode Phasmarhabditis hermaphrodita were applied to soil as an overall spray or as a band spray (8-cm wide), centred on the drill rows (16.7-cm apart). Nematodes were either left undisturbed on the soil surface or harrowed into the soil immediately after application. The control provided by nematodes was compared with that provided by metaldehyde and methiocarb pellets broadcast at the recommended rate immediately after drilling. In this experiment, winter wheat on plots treated with IJs showed significantly less slug damage than on wheat plots treated with metaldehyde or methiocarb pellets or untreated plots. There was no significant difference in plant damage between plots treated with band and overall spray applications of IJs, nor was there any significant difference between plots with and without harrowing. There was also no significant difference between untreated plots and plots treated with metaldehyde or methiocarb pellets, probably because rainfall shortly after treatment rendered the pellets ineffective. In experiment 2, nematodes were applied as an overall spray or plots were not treated with nematodes before soil was cultivated with tines, Roterra or Dutzi cultivators. Nematode application before soil cultivation using tines or Roterra reduced the number of plants damaged significantly. However, nematodes applied before Dutzi cultivation appeared to be rendered ineffective. Damage to winter wheat was lowest in plots that had been sprayed with nematodes and subsequently cultivated with tines or Roterra.     

Susceptibility of indigenous UK earthworms and an invasive pest flatworm to the slug parasitic nematode Phasmarhabditis hermaphrodita

We exposed five earthworm species, one flatworm species and the susceptible slug Deroceras reticulatum to the recommended field application rate and five times this rate of Phasmarhabditis hermaphrodita. P. hermaphrodita caused significant mortality to D. reticulatum at the recommended rate and five times the recommended rate. Survival of the earthworms and the flatworm was not different from the untreated controls. P. hermaphrodita is a safe biological control agent causing no harm to earthworms and could not be used to kill Arthurdendyus triangulatus.     

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.     

The acceptability of meadow plants to the slug Deroceras reticulatum and implications for grassland restoration

Background and Aims

Despite the selective pressure slugs may exert on seedling recruitment there is a lack of information in this context within grassland restoration studies. Selective grazing is influenced by interspecific differences in acceptability. As part of a larger study of how slug–seedling interactions may influence upland hay meadow restoration, an assessment of relative acceptability is made for seedlings of meadow plants to the slug, Deroceras reticulatum.

Methods

Slug feeding damage to seedling monocultures of 23 meadow species and Brassica napus was assessed in microcosms over 14 d. The severity and rate of damage incurred by each plant species was analysed with a generalized additive mixed model. Plant species were then ranked for their relative acceptability.

Key Results

Interspecific variation in relative acceptability suggested seedlings of meadow species form a hierarchy of acceptability to D. reticulatum. The four most acceptable species were Achillea millefolium and the grasses Holcus lanatus, Poa trivialis and Festuca rubra. Trifolium pratense was acceptable to D. reticulatum and was the second highest ranking forb species. The most unacceptable species were mainly forbs associated with the target grassland, and included Geranium sylvaticum, Rumex acetosa, Leontodon hispidus and the grass Anthoxanthum odoratum. A strong positive correlation was found for mean cumulative feeding damage and cumulative seedling mortality at day 14.

Conclusions

Highly unacceptable species to D. reticulatum are unlikely to be selectively grazed by slugs during the seedling recruitment phase, and were predominantly target restoration species. Seedlings of highly acceptable species may be less likely to survive slug herbivory and contribute to seedling recruitment at restoration sites. Selective slug herbivory, influenced by acceptability, may influence community-level processes if seedling recruitment and establishment of key functional species, such as T. pratense is reduced.