Pathogenicity of wild and commercial Phasmarhabditis hermaphrodita exposed to the pestiferous slug Deroceras invadens.

Many terrestrial gastropods are pestiferous and pose a significant threat to agriculture, horticulture and floriculture. They are usually controlled by metaldehyde based pellets but an alternative control method is the slug parasitic nematode Phasmarhabditis hermaphrodita, which has been formulated into a biological control agent (Nemaslug®) for use by farmers and gardeners to kill certain pestiferous slug species in 4-21 days. The current strain of P. hermaphrodita (called DMG0001) has been used in commercial production since 1994, but there is little information about the pathogenicity of wild strains of P. hermaphrodita towards slugs. Here, we exposed the pestiferous slug Deroceras invadens to nine wild isolated strains of P. hermaphrodita (DMG0002, DMG0003, DMG0005, DMG0006, DMG0007, DMG0008, DMG0009, DMG0010 and DMG0011) and the commercial strain (DMG0001) to three doses (0, 500 and 1000 nematodes per ml). Survival and feeding were recorded over 14 days. All wild P. hermaphrodita strains (other than DMG0010) and P. hermaphrodita (DMG0001), applied at 500 nematodes per ml, caused significant mortality to D. invadens compared to an uninfected control. Similarly, all P. hermaphrodita strains applied at 1000 nematodes per ml, caused significant mortality to D. invadens compared to an uninfected control. Overall, all wild P. hermaphrodita strains (other than DMG0011) caused significantly more mortality than P. hermaphrodita DMG0001 at one or both nematode concentrations. In summary, we have found some wild P. hermaphrodita strains were more virulent than P. hermaphrodita (DMG0001). Ultimately, these strains could potentially be developed as alternative, efficient biological control agents for use against slugs.

Photorhabdus: a tale of contrasting interactions.

Different model systems have, over the years, contributed to our current understanding of the molecular mechanisms underpinning the various types of interaction between bacteria and their animal hosts. The genus Photorhabdus comprises Gram-negative insect pathogenic bacteria that are normally found as symbionts that colonize the gut of the infective juvenile stage of soil-dwelling nematodes from the family Heterorhabditis. The nematodes infect susceptible insects and release the bacteria into the insect haemolymph where the bacteria grow, resulting in the death of the insect. At this stage the nematodes feed on the bacterial biomass and, following several rounds of reproduction, the nematodes develop into infective juveniles that leave the insect cadaver in search of new hosts. Therefore Photorhabdus has three distinct and obligate roles to play during this life-cycle: (1) Photorhabdus must kill the insect host; (2) Photorhabdus must be capable of supporting nematode growth and development; and (3) Photorhabdus must be able to colonize the gut of the next generation of infective juveniles before they leave the insect cadaver. In this review I will discuss how genetic analysis has identified key genes involved in mediating, and regulating, the interaction between Photorhabdus and each of its invertebrate hosts. These studies have resulted in the characterization of several new families of toxins and a novel inter-kingdom signalling molecule and have also uncovered an important role for phase variation in the regulation of these different roles.

Diet composition has a differential effect on immune tolerance in insect larvae exposed to Mesorhabditis belari, Enterobacter hormaechei and its metabolites.

In insects, diet plays an important role in growth and development. Insects can vary their diet composition based on their physiological needs. In this study we tested the influence of diet composition involving varying concentrations of macronutrients and zinc on the immune-tolerance following parasite and pathogen exposure in Spodoptera litura larvae. We also tested the insecticidal potential of Mesorhabditis belari, Enterobacter hormaechei and its secondary metabolites on Spodoptera litura larvae. The results shows macronutrient composition does not directly affect the larval tolerance to nematode infection. However, Zinc supplemented diet improved the immune tolerance. While larvae exposed to bacterial infection performed better on carbohydrate rich diet. Secondary metabolites from bacteria produced an immune response in dose dependent mortality. The study shows that the larvae maintained on different diet composition show varied immune tolerance which is based on the type of infection.

A nematode that can manipulate the behaviour of slugs.

The ability of parasites to manipulate the behaviour of their hosts has evolved multiple times, and has a clear fitness benefit to the parasite in terms of facilitating growth, reproduction and transfer to suitable hosts. The mechanisms by which these behavioural changes are induced are poorly understood, but in many cases parasite manipulation of serotonergic signalling in the host brain is implicated. Here we report that Phasmarhabditis hermaphrodita, a parasite of terrestrial gastropod molluscs, can alter the behaviour of slugs. Uninfected slugs (Deroceras panormitanum, Arion subfuscus and Arion hortensis) avoid areas where P. hermaphrodita is present, but slugs infected with P. hermaphrodita are more likely to be found where the nematodes are present. This ability is specific to P. hermaphrodita and other nematodes (Steinernema carpocapsae and Heterorhabditis bacteriophora) do not induce this behavioural change. To investigate how P. hermaphrodita changes slug behaviour we exposed slugs to fluoxetine (a selective serotonin reuptake inhibitor) and cyproheptadine (a serotonin receptor antagonist). Uninfected slugs fed fluoxetine no longer avoided areas where P. hermaphrodita was present; and conversely, infected slugs fed cyproheptadine showed no increased attraction to areas with nematodes. These findings suggest that a possible mechanism by which P. hermaphrodita is able to manipulate parasite avoidance behaviour in host slugs is by manipulating serotonergic signalling in the brain, and that increased serotonin levels are potentially associated with a reduction in parasite avoidance.

Nematodes that associate with terrestrial molluscs as definitive hosts, including Phasmarhabditis hermaphrodita (Rhabditida: Rhabditidae) and its development as a biological molluscicide.

Terrestrial molluscs (Mollusca: Gastropoda) are important economic pests worldwide, causing extensive damage to a variety of crop types, and posing a health risk to both humans and wildlife. Current knowledge indicates that there are eight nematode families that associate with molluscs as definitive hosts, including Agfidae, Alaninematidae, Alloionematidae, Angiostomatidae, Cosmocercidae, Diplogastridae, Mermithidae and Rhabditidae. To date, Phasmarhabditis hermaphrodita (Schneider, 1859) Andrássy, 1983 (Rhabditida: Rhabditidae) is the only nematode that has been developed as a biological molluscicide. The nematode, which was commercially released in 1994 by MicroBio Ltd, Littlehampton, UK (formally Becker Underwood, now BASF) under the tradename Nemaslug®, is now sold in 15 different European countries. This paper reviews nematodes isolated from molluscs, with specially detailed information on the life cycle, host range, commercialization, natural distribution, mass production and field application of P. hermaphrodita.

Quantifying anuran microhabitat use to infer the potential for parasite transmission between invasive cane toads and two species of Australian native frogs.

Parasites that are carried by invasive species can infect native taxa, with devastating consequences. In Australia, invading cane toads (Rhinella marina) carry lungworm parasites (Rhabdias pseudosphaerocephala) that (based on previous laboratory studies) can infect native treefrogs (Litoria caerulea and L. splendida). To assess the potential of parasite transmission from the invader to the native species (and from one infected native frog to another), we used surveys and radiotelemetry to quantify anuran microhabitat use, and proximity to other anurans, in two sites in tropical Australia. Unsurprisingly, treefrogs spent much of their time off the ground (especially by day, and in undisturbed forests) but terrestrial activity was common at night (especially in anthropogenically modified habitats). Microhabitat overlap between cane toads and frogs was generally low, except at night in disturbed areas, whereas overlap between the two frog species was high. The situations of highest overlap, and hence with the greatest danger of parasite transmission, involve aggregations of frogs within crevices by day, and use of open ground by all three anuran species at night. Overall, microhabitat divergence between toads and frogs should reduce, but not eliminate, the transmission of lungworms from invasive toads to vulnerable native frogs.

Interactions between the entomopathogenic nematode Heterorhabditis sonorensis (Nematoda: Heterorhabditidae) and the saprobic fungus Fusarium oxysporum (Ascomycota: Hypocreales).

In this study, we assessed the effect of the saprobic fungus, Fusarium oxysporum (Ascomycota: Hypocreales) on the fitness of the entomopathogenic nematode Heterorhabditis sonorensis (Caborca strain). Sand column assays were considered to evaluate the effect of fungal mycelia on infective juvenile (IJ) movement and host access. Additionally, we investigated the effect of fungal spores on the nematodes' ability to search for a host, its virulence, penetration efficiency and reproduction. Three application timings were considered to assess interactions between the fungus and the nematodes. In vitro assays were also considered to determine the effect of fungal extracts on the nematode's symbiotic bacteria. Our observations indicate that presence and age of fungal mycelia significantly affect IJ movement in the sand columns and their ability to establish in the host. These results were also reflected in a reduced insect mortality. In particular, treatments with the 15 days old mycelia showed a significant reduction in insect mortality and penetration efficiency. Presence of fungal spores also impacted nematode virulence and reproduction. In particular, two of the application timings tested (simultaneous [EPN and fungal spores applied at the same time] and alternate I [EPN applied first, fungus applied 24h later]) resulted in antagonistic interactions. Moreover, IJ progeny was reduced to half in the simultaneous application. In vitro assays revealed that fungal extracts at the highest concentration tested (10mg/ml) inhibited the growth of the symbiotic bacteria. Overall, these results suggest that saprobic fungi may play an important role in regulating. EPN populations in the soil, and that they may be one of the factors that impact nematode survival in the soil and their access to insect hosts.

The potential for controlling Pangaeus bilineatus (Heteroptera: Cydnidae) using a combination of entomopathogens and an insecticide.

The peanut burrower bug, Pangaeus bilineatus (Say), is an important pest of peanut (Arachis hypogaea L.) in the southern United States. Current control methods for this pest, which are based on the use of chemical insecticides, have not been successful. Our objective was to determine if entomopathogens applied alone or in combination with a standard chemical insecticide would provide superior levels of P. bilineatus mortality compared with the standard chemical applied alone. Specifically, we investigated the efficacy of an entomopathogenic nematode, Heterorhabditis bacteriophora Poinar (Oswego strain), and a fungus, Beauveria bassiana (Balsamo) Vuillemin (GHA strain), applied alone or in combination with chlorpyrifos. When applied as single treatments, the two entomopathogens were not pathogenic, that is, they did not cause mortality in P. bilineatus adults that was different from the nontreated control. However, 3 and 7 d posttreatment, the combination of the H. bacteriophora and chlorpyrifos caused higher mortality than the nematode, fungus, or insecticide alone, or the combination of chlorpyrifos and B. bassiana. The nature of the interaction between H. bacteriophora and chlorpyrifos was synergistic, which is of particular interest, given that this is the first time a synergy is being reported between a nematode that was not pathogenic when applied alone and a chemical insecticide. B. bassiana and its combination with the chlorpyrifos did not significantly increase insect mortality compared with chlorpyrifos alone or the control. Based on the observation of synergy, the combination of H. bacteriophora and chlorpyrifos should be investigated further for potential adoption in the management of P. bilineatus.

A novel method for infecting Drosophila adult flies with insect pathogenic nematodes.

Drosophila has been established as an excellent genetic and genomic model to investigate host-pathogen interactions and innate immune defense mechanisms. To date, most information on the Drosophila immune response derives from studies that involve bacterial, fungal or viral pathogens. However, immune reactions to insect parasitic nematodes are still not well characterized. The nematodes Heterorhabditis bacteriophora live in symbiosis with the entomopathogenic bacteria Photorhabdus luminescens, and they are able to invade and kill insects. Interestingly, Heterorhabditis nematodes are viable in the absence of Photorhabdus. Techniques for infecting Drosophila larvae with these nematodes have been previously reported. Here, we have developed a method for infecting Drosophila adult flies with Heterorhabditis nematodes carrying (symbiotic worms) or lacking (axenic worms) their associated bacteria. The protocol we present can be readily adapted for studying parasitic strategies of other insect nematodes using Drosophila as the host infection model.

Genetic diversity and comparative analysis of gene expression between Heterorhabditis bacteriophora Az29 and Az36 isolates: uncovering candidate genes involved in insect pathogenicity.

Entomopathogenic nematode Heterorhabditis bacteriophora Az29 and Az36 isolates with different virulence against Popillia unipuncta and soil survival time were isolated from the Azorean archipelago (Portugal) and used for the study. RAPD analysis revealed a very low-level of genetic diversity (GD(axenic Az36 isolate)(axenic Az29 isolate)=0.2338±0.0541) between axenic Az29 and Az36 isolates, and a relative low-level of diversity (GD(Az36 isolate)(Az29 isolate)=0.3366±0.0471) between Az29 and Az36 isolates. To unravel the molecular differences, a suppressive subtractive hybridization library was constructed from the parasitic stage. Assembling 150 high quality ESTs produced 70 singletons and 17 contigs. BLAST analysis revealed that 48 ESTs showed significant similarity to known protein and 39 ESTs had no significant hits in the database, perhaps representing novel genes. Functional annotation revealed some of these genes to be involved in metabolism, cellular process and signaling, information storage and processing, stress response and host-parasite interactions. Genes with a role in the parasitism process were identified including lectin, metalloprotease, enolase, chitinase, surface-associated antigen, and as well as genes (aquaporin, Hsp70A, Hsp10 and Hsp20) essential for stresses tolerance. The work described here provides the molecular data necessary for investigating the fundamental molecular aspects of host-parasite interactions. Future investigations should be focused on determining the molecular mechanism of those genes in entomopathogenic nematode life cycle.

Effects of associated bacteria on the pathogenicity and reproduction of the insect-parasitic nematode Rhabditis blumi (Nematoda: Rhabditida).

Three bacteria, Alcaligenes faecalis , Flavobacterium sp., and Providencia vermicola , were isolated from dauer juveniles of Rhabditis blumi . The pathogenic effects of the bacteria against 4th instar larvae of Galleria mellonella were investigated. Providencia vermicola and Flavobacterium sp. showed 100% mortality at 48 h after haemocoelic injection, whereas A. faecalis showed less than 30% mortality. Dauer juveniles showed 100% mortality against G. mellonella larvae, whereas axenic juveniles, which do not harbor associated bacteria, exhibited little mortality. All of the associated bacteria were used as a food source for nematode growth, and nematode yield differed with bacterial species. Among the bacterial species, P. vermicola was most valued for nematode yield, showing the highest yield of 5.2 × 10(4) nematodes/mL in the plate. In bacterial cocultures using two of the three associated bacteria, one kind stimulated the other. The highest total bacterial yield of 12.6 g/L was obtained when the inoculum ratio of P. vermicola to A. faecalis was 10:1. In air-lift bioreactors, the nematode growth rate increased with an increasing level of dissolved oxygen. The maximum nematode yield of 1.75 × 10(5) nematodes/mL was obtained at 192 h with an aeration rate of 6 vvm.

Probing the tri-trophic interaction between insects, nematodes and Photorhabdus.

SUMMARY: Photorhabdus sp. are entomopathogenic bacteria which, upon experimental infection, interact with the insect immune system, but little is known about the roles of their symbiotic nematode partners Heterorhabditis sp. in natural infections. Here, we investigated the respective contributions of nematodes and bacteria by examining humoral and cellular immune reactions of the model lepidopteran insect Manduca sexta against Heterorhabditis carrying Photorhabdus, nematodes free of bacteria (axenic nematodes) and bacteria alone. Insect mortality was slower following infection with axenic nematodes than when insects were infected with nematodes containing Photorhabdus, or the bacteria alone. Nematodes elicited host immune responses to a lesser extent than bacteria. Transcription of certain recognition and antibacterial genes was lower when insects were naturally infected with nematodes carrying no bacteria compared to insects that received bacteria, either with or without nematodes. Axenic nematodes also did not elicit such high levels of phenoloxidase activity and haemocyte aggregates as did treatments involving Photorhabdus. By contrast, the phagocytic capability of host haemocytes was decreased by both axenic and bacteria-associated nematodes, but not by Photorhabdus alone. These results imply that both bacteria and nematodes contribute separately to the pathogenic modulation of host immune responses during natural infections by the mutualistic Heterorhabdus-Photorhabdus complex.

Heterorhabditis amazonensis (Rhabditidae: Heterorhabditidae), strain RSC-5, for biological control of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The aim of this study was to evaluate the influence of different doses of Heterorhabditis amazonensis RSC-5 on the biological parameters of engorged females of Rhipicephalus (Boophilus) microplus. The female ticks, individually identified, were divided into six groups of 20 each and exposed to the following nematode concentrations: 0, 75, 150, 300, 600, and 1,200/female. The following parameters were observed: initial weight, final weight, alteration weight, egg mass weight, pre-oviposition period, oviposition period, survival period, incubation period, hatching percentage, reproductive efficiency index (REI), nutritional efficiency index (NI), and percentage of control. There were no significant differences observed for the initial weight, pre-oviposition period, incubation period, and NI (p > 0.05) between the various treated groups and the control group. However, there were significant differences (p < 0.01) for the oviposition and survival periods between all the treated groups and the control group. For the final weight, alteration weight, egg mass weight, and REI, only the treatment with 1,200 nematode/female did not differ significantly from the respective figures for the control group (p > 0.05). The highest control rate was 67.8%, obtained in the treatment with a concentration of 300 entomopathogenic nematodes/female. The results demonstrate that this nematode has a deleterious effect on the majority of the biological parameters analyzed.

Rates of development in male and female Wood Frogs and patterns of parasitism by lung nematodes.

Researchers are becoming interested in testing whether investment in growth and/or development trades off against investment in parasite defence. We tested this idea by examining relations between development of Wood Frogs (Rana sylvatica) and susceptibility to lung nematodes (Rhabdias ranae). Male and female frogs reared in outdoor mesocosms were the same length and mass at metamorphosis. However, males metamorphosed sooner than females. Lung nematodes were no more likely to penetrate male versus female metamorphs following controlled exposures, but males had higher intensities of adult female worms and the largest worms per host were, on average, of larger size in male metamorphs. Males that took longer to metamorphose carried higher numbers of worms in their lungs than males that metamorphosed early. In comparison, females that developed faster harboured more worms in their lungs than females that took longer to reach metamorphosis. Our results suggest that variation in susceptibility to lung nematodes is influenced by host sex and possibly also by sex-specific relations with developmental rate. Further, male hosts might prove to be a more important source of infective stages of worms than female hosts.

Effect of the change in energy reserves on the entomopathogenic nematode efficacy.

The free-living infective juveniles of the entomopathogenic nematodes are non-feeding, so the stored energy reserves in these juveniles are of great importance. The relationship between the energy reserves and the efficacy of the entomopathogenic nematodes of genera Heterorhabditis and Steinernema was studied. New progenies of S. riobrave and H. bacteriophora (ISK-2 strains), were obtained using the continuous culturing method of nematode juveniles (IJs), for the several cycles under the optimum condition of the temperature (25 degrees C) and nematode density (20 IJs/ larva), inside the host Galleria mellonella. Thus, the nematode efficacy was maximized with a high conservation of energy reserves. The results showed that there was an increase in the penetration rate and the virulence through the new progenies of S. riobrave and H. bacteriophora than in the original progenies, with an increase in the energy reserves.

Biological control of terrestrial molluscs using Phasmarhabditis hermaphrodita--progress and prospects.

Phasmarhabditis hermaphrodita Schneider (Nematoda: Rhabditidae) is a nematode that parasitises a wide range of slug and snail species. It has been formulated into a biological control agent (Nemaslug) and was commercialised in 1994. It is now available in fourteen European countries. A review is given of all research on P. hermaphrodita, including basic biology, mass cultivation, formulation, host range, application strategies, field efficacy and effects on non-target organisms. The many critical gaps in present knowledge are highlighted, and future research is proposed that will lead to greater understanding of this unusual parasite and may enable its more widespread use in the management of mollusc pests.

Interactions between isolates of the entomopathogenic fungus Metarhizium anisopliae and the entomopathogenic nematode Heterorhabditis bacteriophora JPM4 during infection of the sugar cane borer Diatraea saccharalis (Lepidoptera: Pyralidae).

Interactions between the nematode Heterorhabditis bacteriophora isolate JPM4 and the fungus Metarhizium anisopliae, isolates LPP45 and LPP39, were studied during dual infections of Diatraea saccharalis. Mortality, production of infective juveniles (IJs) and production of conidia were evaluated. A positive effect was demonstrated for host mortality in duel infections of JPM4 and LPP39, causing 100% mortality with LT(50) and LT(95) values of 1.8 and 2.8 days, respectively. Higher values were seen when using the nematode or fungi individually. However, a combination of JPM4+LPP39 caused a significant reduction in IJ production. The results show that faster time to death, a moderately virulent fungal isolate could be combined with the nematode, however at the expense of IJ production.

Pelodera (syn. Rhabditis) strongyloides as a cause of dermatitis--a report of 11 dogs from Finland.

BACKGROUND: Pelodera (Rhabditis) strongyloides is a small saprophytic nematode that lives in decaying organic matter. On rare occasions, it can invade the mammalian skin, causing a pruritic, erythematous, alopecic and crusting dermatitis on skin sites that come into contact with the ground. Diagnosis of the disease is based on case history (a dog living outdoors on damp straw bedding) with characteristic skin lesions and on the demonstration of typical larvae in skin scrapings or biopsy. Pelodera (rhabditic) dermatitis cases have been reported mainly from Central European countries and the United States. CASE PRESENTATION: During 1975-1999, we verified 11 canine cases of Pelodera dermatitis in Finland. The cases were confirmed by identifying Pelodera larvae in scrapings. Biopsies for histopathology were obtained from three cases, and typical histopathological lesions (epidermal hyperplasia, epidermal and follicular hyperkeratosis, folliculitis and furunculosis with large numbers of nematode larvae of 25-40 microm of diameter within hair follicles) were present. The Pelodera strongyloides dermatitica strain from the first verified case in Finland has been maintained in ordinary blood agar in our laboratory since 1975. Light microscopy (LM) and scanning electron microscopy (SEM) studies were employed to obtain detailed morphological information about the causative agent. The rhabditiform oesophagus at all developmental stages, the morphology of the anterior end of the nematode, copulatory bursa and spicules of the male and the tail of the female were the most important morphological features for identifying P. strongyloides. CONCLUSION: These cases show that Pelodera dermatitis occurs in Finland, and also farther north than described earlier in the literature. This condition should be considered when a dog living outdoors has typical skin lesions situated at sites in contact with the ground as the main presenting clinical feature. The fastest and easiest way to confirm the diagnosis is to demonstrate typical larvae in skin scrapings. In uncertain cases, skin biopsy and culturing of the worms are recommended as supplementary diagnostic procedures.

The exbD gene of Photorhabdus temperata is required for full virulence in insects and symbiosis with the nematode Heterorhabditis.

Photorhabdus are bacteria found colonizing the gut of a specialized stage of the nematode Heterorhabditis, called the infective juvenile (IJ). The IJ is a free-living stage of the nematode that seeks out and infects insect larvae. Once inside the insect the IJ release Photorhabdus into the haemolymph where the bacteria rapidly proliferate, killing the insect within 48-72 h. The nematodes grow and reproduce in the insect cadaver by feeding on the Photorhabdus biomass. In this study we use Photorhabdus temperata K122 to show that genes involved in iron acquisition play a key role during the course of the tripartite bacteria-nematode-insect interaction. We show that a strain carrying a mutation in a gene with homology to exbD, encoding a component of the TonB complex, is unable to grow well in conditions where iron is not freely available. In addition, this mutant, BMM417, requires a longer time to kill the insect larvae than the wild-type bacteria and this defect in pathogenicity is complemented by the co-injection of iron. Moreover, the increase in LT(50) observed with BMM417 is correlated with a significantly slower in vivo growth rate suggesting that iron is limiting in the insect. We also show that BMM417 is unable to support the growth and development of the Heterorhabditis nematode. Addition of exogenous iron to the growth media restores nematode growth and development on BMM417, suggesting that aspects of iron metaboism in Photorhabdus are important during the symbiosis with the nematode.