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Background Culicoides insignis is a confirmed vector of bluetongue virus (BTV) throughout the American tropics and a possible vector of epizootic hemorrhagic disease virus (EHDV) in Florida. Despite its importance, fundamental information on the biology and ecology of this vector species is lacking. In this study, we examined the oviposition of C. insignis under laboratory conditions, monitored the development of immature stages and attempted colonization of this species. Methods Live C. insignis females were collected from the field using CDC-UV-LED traps, allowed to blood-feed on live chicken and given various natural substrates for oviposition in two-choice assays. The eggs deposited were transferred to 0.3% agar slants, and the hatched larvae were provided a diet of Panagrellus redivivus Linnaeus nematodes and the development of all immature stages was monitored. Results Culicoides insignis females exhibited an overall oviposition preference for dishes containing mud from their larval habitat as gravid females deposited a significantly higher number of eggs on these dishes (35.3 eggs/female) than on controls (17.7 eggs/female). The ovipositing females also deposited a higher percentage of eggs on substrates with habitat mud and other organically enriched muds (≥ 75.2%) compared to controls (31.0%). The larvae developed successfully to adulthood on the nematode diet, exhibiting high overall larval survival rates (85.0%). Sex ratios of the F1 generation were male biased, approximately 3:1 (male:female). Captive mating could not be induced in the F1 adults. Conclusions Mud from the larval habitat and other organically enriched muds provide strong oviposition cues to C. insignis under laboratory conditions. Further studies will be needed to identify the key biotic/abiotic factors influencing midge oviposition in the field. The agar/nematode method is effective for the rearing of C. insignis larvae. However, further studies will be needed to address the issue of male-biased sex ratios in the progeny and to examine the mating habits/cues of C. insignis in nature, which may provide clues towards inducing captive mating in the F1 adults. Graphical abstract

To better understand the nanosize-relevant toxic effects and underlying mechanisms, N-acetylcysteine (NAC), as a mitigation agent, an ionic form of Zn (ZnCl2), and the binary mixture of ZnO with different particle sizes (15 nm and 140 nm), was used in toxicity assays with the nematode Panagrellus redivivus. The ZnCl2 concentrations were applied to show the amount of dissolved Zn ions present in the test system. Reactive oxygen species (ROS) measuring method was developed to fit the used test system. Our studies have shown that NAC can mitigate the toxic effects of both studied particle sizes. In the applied concentrations, ZnCl2 was less toxic than both of the ZnO particles. This finding indicates that not only ions and ROS produced by the dissolution are behind the toxic effects of the ZnO NPs, but also other particle size-dependent toxic effects, like the spontaneous ROS generation, are also relevant. When the two materials were applied in binary mixtures, the toxic effects increased significantly, and the dissolved zinc content and the ROS generation also increased. It is assumed that the chemical and physical properties of the materials have been mutually reinforcing to form a more reactive mixture that is more toxic to the P. redivivus test organism. Our findings demonstrate the importance of using mitigation agent and mixtures to evaluate the size-dependent toxicity of the ZnO. Graphical abstract

Bacteria with nematicidal effects have attracted interest as biocontrol agents of pests and diseases due to their relatively low environmental impact compared to chemical management strategies1. The damage they cause to target nematodes is diverse and imperfectly understood4.Photorhabdus spp. (Enterobacteriaceae) lives symbiotically within the nematode Heterorhabditis spp. (Rhabditida: Heterorhabditidae). It is known that secondary metabolites of this gram-negative bacterium exhibit insecticidal activity, mainly in Lepidopteran and Coleopteran insects2. Studies have also shown that cell-free culture filtrates of Photorhabdus spp. have nematicidal activity on free-living nematodes and pest phytonematodes; however, the causes of death in these targets are still being studied. Fil: Salas, Augusto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Achinelly, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; Argentina Fil: Sauka, Diego Herman. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Nematode-trapping fungi constitute a guild of species that develop specialized trapping structures with which they capture and digest nematodes. These traps are not constitutively present, but instead develop in the presence of nematodes ; they attract them , adhere to their cuticle and eventually pierce it, extending hyphae inside their bodies. Fil: Maestro, Mariano. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Sauka, Diego Herman. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina

Free-living nematode, Panagrellus redivivus, was tested as live food for blue swimming crab, Portunus pelagicus larvae during the first feeding stage. A series of experiments were conducted to determine the acceptability of the free-living nematodes in P. pelagicus larvae at first feeding. The optimum nematode density and the response of the larvae to nutritionally enriched nematode were also been examined. Newly hatched P. pelagicus, distributed into each 50 mL experiment plastic containers contains 30 individual P. pelagicus larvae. Microworms will be given four times day−1 at around 0800 h, 1100 h, 1400 h and 1600 h after the water exchange. Duration of feeding experiments was up to day 10–15 and results show that their survival rate of zoea 1: 56%, zoea 2: 74%, zoea: 3 84% and zoea 4: 66%. The results also showed that P. redivivus exhibited better Specific Growth Rate from Zoea 2 to Zoea 3 stage compared to the Artemia based diets. Observations indicated that the P. pelagicus, larvae readily fed on free-living nematodes as early as 2 days post-hatching, the start of exogenous feeding. Studies focusing on the alternative live food at the early larval stages can improve culture information for future and mass aquaculture commercialization of portunid crabs. Keywords: Aquaculture, Crustacean, Early feeding, Nursery culture, Seed production

Nematode-trapping fungi constitute a guild of species that develop specialized trapping structures with which they capture and digest nematodes. These traps are not constitutively present, but instead develop in the presence of nematodes3; they attract them2, adhere to their cuticle and eventually pierce it, extending hyphae inside their bodies. Instituto de Microbiología y Zoología Agrícola (IMYZA) Fil: Maestro, Mariano. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentina Fil: Sauka, Diego Hernan. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentina

Synadenium grantii is a Euphorbiaceae plant commonly found in Brazil, known as Janaúba or Leitosinha, whose latex is traditionally used for several purposes. However, it is not known whether the nematicidal action of this plant latex occurs due to the action of proteases. The present work aims to evaluate the nematicidal activity of proteases from Synadenium grantii latex on Meloidogyne incognita and Panagrellus redivivus. S. grantii latex used in the present study was collected from specimens found in Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The drained latex was collected in Eppendorf microtubes and immediately stored on ice at 4 °C. After this extraction, the latex was frozen (-20 °C) during 2 hours, thawed at room temperature (25 °C) and centrifuged at 10,000 g at 4 °C for 30 minutes to remove larger particles and concentrate the proteases. After the centrifugation, assays of enzymatic activity were performed in order to know in which of the phases the enzymes were found. S. grantii latex presented protease, but no chitinase activity. The results show that there was a significant difference (p

This study was aimed to evaluate the in vitro lethal activity of the nematophagous fungi Clonostachys rosea against 5 nematodes species belonging to different taxa. Two groups of 35 Petri dishes (PD) each were divided into 5 series of 7 (PD). Group 1 (series 1, 2, 3, 4, and 5) contained only water agar; meanwhile group 2 plates (series 6, 7, 8, 9, and 10) contained C. rosea cultures growth on water agar. Every plate from the two groups was added with 500 nematodes corresponding to the following genera/specie: Haemonchus contortus, Caenorhabditis elegans, Rhabditis sp., Panagrellus redivivus, and Butlerius sp. After 5-day incubation at room temperature, free (nontrapped) larvae were recovered from plates using the Baermann funnel technique. Recovered nematodes were counted and compared with their proper controls. Results shown an important reduction percentage of the nematode population attributed to the fungal lethal activity as follows: H. contortus (L3) 87.7%; C. elegans 94.7%; Rhabditis sp. 71.9%; P. redivivus 92.7%; and Butlerius sp. 100% (p≤0.05). The activity showed by C. rosea against the H. contortus can be crucial for further studies focused to the biological control of sheep haemonchosis, although the environmental impact against beneficial nematodes should be evaluated.

Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli . The binary toxin also shows strong nematicidal activity against three other free-living nematodes ( Pristionchus pacificus , Panagrellus redivivus , and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction. IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes ( Pristionchus pacificus , Panagrellus redivivus , and Acrobeloides sp.). The two subunits, Pp-ANP1a and Pp-ANP2a, can be expressed separately and reconstituted to form the active complex. Pp-ANP shows some distinct characteristics compared with other toxins, including Escherichia coli enterotoxin and cholera toxin. The present study indicates that Pp-ANP is a novel binary toxin and that it has potential applications in controlling parasitic nematodes and in studying toxin-host interaction.

This study evaluates the potential application of silver nanoparticles (AgNPs) as antimicrobial or nematicidal agents produced by the extremophile Bacillus pumilus, which was isolated from the alkaline Wadi El-Natrun Lake in Egypt. The AgNPs were characterized by ultraviolet–visible absorption spectroscopy, transmission electron microscopy, and energy dispersive x-ray spectroscopy. The size of AgNPs formed ranged from 20.12 to 29.48 nm. Panagrellus redivivus was exposed to different concentrations (0, 50, 100, 150, and 200 µg/mL) of AgNPs in a 5 mL nematode suspension (1×103 mL-1). The best result occurred at AgNP concentrations of 150 and 200 µg/mL, with death rates of 80% and 91%, respectively, following 48 h of exposure. AgNPs also exhibited potent antimicrobial properties when using Gram-negative and Gram-positive human pathogens, with MIC and MBC values of 5 μg/mL and 10 μg/mL, respectively. These laboratory assays prove that biologically synthesized AgNPs are an ecofriendly material that can be used in lieu of solvents or toxic chemicals.

Made available in DSpace on 2018-12-11T16:39:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-10-02 The distribution of total and nematophagous fungi and nematodes is influenced by different plant species and environmental factors. The effect of moisture content and soil organic matter (SOM) in the frequency of fungi and nematodes was examined in Eutrustox soil with lettuce (Lactuca sativa), banana (Musa cavendishii), impatiens (Impatiens walleriana) and bahiagrass (Paspalum notatum). Total fungi and nematophagous counts were made in the rhizosphere soil (RS) and non-rhizosphere soil (NRS). The fungi were isolated from each soil, and their ability to predate Panagrellus redivivus nematode was evaluated. The fungi frequency in the RS and NRS ranged from 6.9 to 25.6 � 105 and from 6.9 to 31.2 � 105 CFU g-1 soil, respectively, and nematophagous fungi accounted for 21-34% and 16-33% of total fungi, respectively. The total and nematophagous fungi counts of RS decreased in the order lettuce>banana>bahiagrass>impatiens and lettuce>impatiens>banana>bahiagrass, respectively. Both total and nematophagous fungi counts from NRS decreased in the order lettuce>banana>bahiagrass>impatiens. A positive and significant correlation showed that fungi counts were influenced by the SOM and moisture contents. The abundance of nematodes was influenced by plant species, ranging from 0 to 4.0 � 103 100 g-1 dry soil or roots. It can be concluded that the distribution of total and nematophagous fungi, and nematodes relates to plant species and the SOM and moisture contents. Program of Agropecuary Microbiology Universidade Estadual Paulista (UNESP) Department of Crop Production Universidade Estadual Paulista (UNESP) Program of Agropecuary Microbiology Universidade Estadual Paulista (UNESP) Department of Crop Production Universidade Estadual Paulista (UNESP)

Conidial traps (Ct) in Arthrobatrys oligospora are adhesive structures formed directly on germination of conidia, without an intermediate hyphal phase. A conidial trap-producing mutant was isolated by a procedure analogous to that used for microcycle conidiation. This Ct-strain produced strict conidial traps in abundance on conidia when still on upright conidiophores. The phenotypic stability of the Ct-strain lasted over more than 20 transfers on trap-inducing media. Growth on non-inducing media (5 transfers) and subsequent inoculation on trap-inducing medium again showed traps on conidia on upright conidiophores. The conidial traps attached to nematodes before invasion and digestion. At the subcellular level the Ct were characterized by the presence of many electron-dense microbodies. In addition, in the Ct-strain normal trap formation on the mycelium was considerably increased compared to the parent strain. This high trap-forming ability will make it a useful tool both in rhizosphere studies and in biochemical/molecular investigations.

The infection of nematodes by the endoparasitic fungus Verticillium balanoides was studied by means of optical and electron microscopical techniques. Conidia of this fungus adhere randomly in high numbers to the entire cuticle surface of the nematode Panagrellus redivivus. Adhesion is accomplished by means of a tri-layered adhesive pad located at the apical end of the conidium, the outermost layer of which shows a radiated substructure. After attachment an appressorium which grows through the adhesive pad and establishes a firm contact between the fungal cell wall and the nematode cuticle is formed. Penetration of the cuticle by means of a hyphal outgrowth on the appressorium is followed by the formation of an infection bulb from which trophic hyphae develop that invade the nematode. Approximately 60 h after infection conidiophores develop outside the nematode, producing numerous conidia. © 1993, British Mycological Society. All rights reserved.

We reported recently a unique fungal structure, called the spiny ball, on the vegetative hyphae of Coprinus comatus (O. F. Müll.:Fr.) Pers. Although some observations regarding the role of this structure were presented, its function remained largely unknown. In this study, we showed that purified (isolated and washed) spiny balls could immobilize and kill the free-living nematode Panagrellus redivivus Goodey highly efficiently. Scanning electron microscopy studies illustrated that the spiny structure damaged the nematode cuticle, suggesting the presence of a mechanical force during the process of nematode immobilization. Severe injuries on nematode cuticles caused the leakage of inner materials of the nematodes. When these structures were ground in liquid nitrogen, their killing efficacy against nematodes was lost, indicating that the shape and the complete structure of the spiny balls are indispensable for their function. However, extraction with organic solvents never lowered their activity against P. redivivus , and the extracts showed no obvious effect on the nematode. We also investigated whether C. comatus was able to produce toxins which would aid in the immobilization of nematodes. In total, we identified seven toxins from C. comatus that showed activity to immobilize the nematodes P. redivivus and Meloidogyne incognita (Kofoid et White) Chitwood. The chemical structures of these toxins were identified with nuclear magnetic resonance, mass spectrometry, infrared, and UV spectrum analysis. Two compounds were found to be novel. The toxins found in C. comatus are O-containing heterocyclic compounds.

Efficient killing of nematodes by Stropharia rugosoannulata Farlow ex Murrill cultures was observed. This fungus showed the ability to immobilize the free-living nematode Panagrellus redivivus Goodey within minutes and to immobilize the pine wilt nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle within hours on agar plates. Moreover, P. redivivus worms were completely degraded by the fungus within 24 to 48 h. The cultures of S. rugosoannulata studied shared the characteristic of abundantly producing cells with finger-like projections called acanthocytes. We showed that the nematode-attacking activity of this fungus is carried out by these spiny acanthocytes and that mechanical force is an important factor in the process. Furthermore, the growth and nematode-attacking activity of the fungus in soil were also determined, and our results suggest that acanthocytes are functional in soil.

In a PCR with degenerate primers encoding highly conserved amino acids within ornithine decarboxylases (ODCs) of several organisms, a fragment of the ODC gene of the free-living nematode Panagrellus redivivus was isolated. Northern blot analysis revealed a single 1.7 kb transcript in a mixed-stage population of animals. From this RNA source, a cDNA library was constructed and screened with the PCR fragment. Several cDNA clones were isolated, one of which encodes the complete 435-amino-acid ODC enzyme with a calculated molecular mass of 47.1 kDa. The P. redivivus ODC possesses 126 of the 136 highly conserved amino acids in the enzymes from fungi, invertebrates and vertebrates. Functional amino acids are conserved, suggesting that the two active sites of the P. redivivus ODC are formed at the interface of a homodimer, as described for mammalian ODCs.

Individuals of the free-living nematode Panagrellus redivivus were infected with conidia of the endoparasitic nematophagous fungus Drechmeria coniospora. At different stages of infection the nematodes were added to an agar-plate culture of the predatory fungus Arthrobotrys oligospora which contained traps. Initially, nematodes infected with D. coniospora were invariably captured, penetrated and colonized by A. oligospora. Then both fungi used the nematode contents for development of hyphal biomass outside the nematode. But inside nematodes in which infection by D. coniospora was advanced development of A. oligospora was markedly hampered. In dual-infected nematodes (colonized from opposite ends), the hyphae of A. oligospora were often observed to be dead or degenerate when in close proximity to living mycelium of D. coniospora, indicating that the latter acts as an antagonist against A. oligospora under these conditions. The interaction between trophic hyphae of D. coniospora and A. oligospora involved thickening of the cell wall of A. oligospora at the sites of contact and degradation of the hyphal compartments and shrivelling of the cell wall of A. oligospora. The observed interactions are discussed in relation to mycoparasitism and possible ecological function. © 1994, British Mycological Society. All rights reserved.

The free-living nematode Panagrellus redivivus was found to have relatively high cytosolic glutathione transferase activity. Chromatofocusing indicated that at least four GSH transferase forms were present in the nematode cytosol. An endogenous molecular factor interfered with the binding of the cytosolic GSH transferases to a glutathione affinity matrix and binding only occurred after a partial purification step. The major resolved GSH transferase form B was a basic enzyme that showed no biochemical homology to the three mammalian multigene GSH transferase families and appeared to interact with plant phenols, possible natural substrates. A minor GSH transferase form AT showed a biochemical relationship to the mammalian α family including catalytic activity with a model lipid hydroperoxide substrate.

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