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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

To complete their life cycle, pathogenic nematode-bacteria complexes deter scavengers from feeding on their host cadaver

The life cycle of commercially used molluscicidal rhabditid nematodes Phasmarhabditis hermaphrodita and entomopathogenic steinernematid nematodes is similar: infective stages carry symbiotic bacteria, which kill their host. Nematodes complete their life cycle feeding on the proliferating symbiont and the host tissue. After 1-2 weeks, new infective stages carrying the bacteria leave the host cadaver in search of new hosts. The removal of invertebrate cadavers by scavengers is extremely fast and represents a severe threat to the developing nematodes.Two-choice trials were used to assess prey choice of the generalist predator/scavenger Pterostichus melanarius (Coleoptera: Carabidae) between Deroceras reticulatum (Mollusca: Agriolimacidae) slugs or wax moth Galleria mellonella (Lepidoptera: Pyralidae) larvae killed by infection of P. hermaphrodita/Steinernema affine and control killed by freezing. We demonstrate that the presence of either of the two nematodes tested deters the beetles from consuming infected cadavers. As P. hermaprodita cannot infect an insect host, we hypothesise the deterrent effect being an evolutionary adaptation of the nematode/bacteria complex rather than the ability of the beetles to avoid potentially infective cadavers.     

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.     

Parasitism of Molluscs by Nematodes: Types of Associations and Evolutionary Trends

Although there are no confirmed fossil records of mollusc parasitic nematodes, diverse associations of more than 108 described nematode species with slugs and snails provide a fertile ground for speculation of how mollusc parasitism evolved in nematodes. Current phylogenic resolution suggests that molluscs have been independently acquired as hosts on a number of occasions. However, molluscs are significant as hosts for only two major groups of nematodes: as intermediate hosts for metastrongyloids and as definitive hosts for a number of rhabditids. Of the 61 species of nematodes known to use molluscs as intermediate hosts, 49 belong to Metastrongyloidea (Order Strongylida); of the 47 species of nematodes that use molluscs as definitive hosts, 33 belong to the Order Rhabditida. Recent phylogenetic hypotheses have been unable to resolve whether metastrongyloids are sister taxa to those rhabditids that use molluscs as definitive hosts. Although most rhabditid nematodes have been reported not to kill their mollusc hosts prior to their reproduction, some species are pathogenic. In fact, infective juveniles of Phasmarhabditis hermaphrodita vector a lethal bacterium into the slug host in which they reproduce. This life cycle is remarkably similar to the entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae. Also, the discoveries of Alloionema and Pellioditis in slugs are interesting, as these species have been speculated to represent the ancestral forms of the entomopathogenic nematodes. Development of the infective stage appears to be an important step toward the acquisition of molluscs as definitive hosts, and the association with specific bacteria may have arisen in conjunction with the evolution of necromeny.     

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.     

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.     

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.     

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.     

Mini‐plot field experiments using the rhabditid nematode Phasmarhabditis hermaphrodite for biocontrol of slugs

The nematode Phasmarhabditis hermaphrodita, a parasite of slugs, was cultured in vitro and applied as a drench in two outdoor mini‐plot field experiments to test the capacity of the nematode to protect Chinese cabbage seedlings and wheat seeds from damage by the field slug Deroceras reticulatum. The first experiment compared a single dose of nematodes (2 X 10¹⁰ ha^‐1) with methiocarb pellets added at the recommended field rate (5.5 kg.product ha^‐1 ) and untreated plots. Plots treated with either nematodes or methiocarb pellets had significantly less slug damage than untreated plots and, from the third week onwards, there was significantly less slug damage on plots treated with nematodes than on methiocarb‐treated plots. At the end of the experiment, 6 weeks after treatment, both slug numbers and biomass were significantly higher in untreated plots than in either methiocarb‐treated or nematode‐treated plots. In the second experiment, six nematode doses ranging from 1 X 10 ⁸ to 2 X 10¹⁰ ha^‐1 were compared with a standard rate application of methiocarb pellets and untreated plots. Plant protection improved with increasing nematode dose between 1 X10⁸ and 8 X 10⁸ha^‐1, but showed little or no further improvement at higher doses. Plant protection similar to that given by methiocarb pellets was provided by nematode doses of 8 X 10⁸ ha^‐1 and above.     

Influences of preceding cover crops on slug damage and biological control using Phasmarhabditis hermaphrodita

In a replicated field experiment, ryegrass, vetch and red clover were grown or the soil was kept bare over a 2–month period in summer to compare the effects of these treatments on slug damage to the following crop (Chinese cabbage) and on the efficacy of nematodes (Phasmarhabditis hermaphrodita) applied as biological control agents to the soil at planting time to protect this crop. Slug damage was significantly (c. two times) greater after red clover or vetch than after ryegrass. Damage on plots without cover crop was intermediate and not significantly different from either extreme. Slug damage was reduced by about one‐third by the nematode treatment. The preceding cover crop did not influence nematode efficacy. Numbers of slugs on harvested plants (mainly Deroceras reticulatum and Deroceras panormitanum) were influenced by an interaction between cover crop and nematode treatment. On subplots without nematodes, more slugs were recorded with than without a preceding cover crop. No such differences were found on nematode‐treated subplots. Soil samples were collected at intervals from 0–99 days after nematode treatment to monitor nematode survival and infectivity in bioassays with D. reticulatum. No significant effects of cover crops were detected in bioassays. Moreover, there were no significant effects of nematodes on slug survival. Their effects on slug food consumption were mostly insignificant and any effects were transient and not consistent. However, significantly more slug cadavers contained nematodes when slugs were exposed to nematode‐treated soil. The implications of these results are discussed.     

Physical properties of liquid nematode cultures and the design of recovery operations

Production of nematode-based pesticides involves the recovery of a viable nematode life stage known as the infective juvenile (IJ) from fermentation broth. In this paper we report the physical properties of mature liquid nematode cultures of P. hermaphrodita, S. feltiae and H. megidis. Properties determined were composition, IJ `shear' sensitivity, viscosity, particle size and component density. These measurements were then used to identify potential recovery procedures. Waste components in cultures included non-IJ life stages, dead nematodes, nematode debris, spent media and the nematodes' associated bacteria. Infective juveniles were very sensitive to `shear' compared to baker's yeast. The choice of recovery equipment will therefore be limited to that which produces a low level of stress. Comparison of IJ properties with those of waste components showed that differences in component size, density and settling rate can be used as a basis for separating fermentation waste. Predictions of IJ settling velocity using Stokes' Law and by experiment confirmed that IJs will need to be separated from culture liquid by centrifugation as opposed to gravity settling. The comparison of nematodes revealed a dependence of culture properties on species. This observation suggested that a flexible processing scheme will be required if different species are to be recovered using the same process equipment.