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.     

Feeding Activity and Survival of Slugs,Deroceras reticulatum,Exposed to the Rhabditid Nematode,Phasmarhabditis hermaphrodita: A Model of Dose Response

The slug, Deroceras reticulatum (Stylommatophora: Limacidae), was exposed to different concentrations of infective dauer juveniles of the rhabditid nematode Phasmarhabditis hermaphrodita, in a two-stage bioassay, at 10°C. Slugs were exposed in groups of 10 or 12 to nematodes in plastic boxes filled with soil aggregates for 3 or 5 days and then transferred individually to petri dishes each containing a disk of Chinese cabbage leaf as food. Subsequently, slug food consumption and survival were measured for 10 to 13 days. Models were developed to describe the way that exposure to the nematode caused inhibition of slug feeding followed by death. Both effects were related to nematode concentrations and time after exposure to the nematode. Following exposure to high concentrations (300,000 dauer juveniles per box), slugs were killed rapidly, within a few days after the end of the exposure period. Following exposure to low concentrations of nematodes (7000 or 15,000 per box), substantial numbers of slugs survived until the end of the bioassay, but feeding activity by these slugs was strongly inhibited. It is suggested that inhibition of slug feeding is important for the success of this nematode as a biocontrol agent.     

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.     

Non-Susceptibility of Earthworm Eisenia fetida to the Rhabditid Nematode Phasmarhabditis hermaphrodita, a Biocontrol Agent of Slugs

The rhabditid nematode Phasmarhabditis hermaphrodita is a lethal parasite for slugs that is sold commercially in Europe under the trade name Nemaslug™. We evaluated the effects of P. hermaphrodita on the earthworm Eisenia fetida (Savigny). Adults of E. fetida were exposed in one-liter glass beakers to P. hermaphrodita at three concentrations (1×, 10× and 50× of the field recommended rate of 3×10⁹ billion nematodes/ha) during a 14-day period in an artificial soil substrate. The average body weight, burrowing behavior, mortality and other clinical signals of the earthworms were recorded at 0, 7, and 14 days after exposure to the treatments. In addition, injured earthworms (posterior ends removed) were exposed to the 10× field recommended rate of the commercial formulation. Neither intact nor injured E. fetida showed susceptibility to the slug-parasitic nematode P. hermaphrodita during the 14 days of exposure even at concentrations 10 and 50 times higher than the label dose. However, the worms in the attenuated control (autoclaved formulation) had higher mortality and lost less weight compared to the other treatments. Under the conditions of the test, we conclude that the use of the commercially available strain of P. hermaphrodita is safe to E. fetida.     

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 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.     

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.     

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.