Host cadavers protect entomopathogenic nematodes during freezing

The entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema glaseri, and Steinernema feltiae were exposed to freezing while inside their hosts. Survival was assessed by observing live and dead nematodes inside cadavers and by counting the infective juveniles (IJs) that emerged after freezing. We (1) measured the effects of 24h of freezing at different times throughout the course of an infection, (2) determined the duration of freezing entomopathogenic nematodes could survive, (3) determined species differences in freezing survival. Highest stage-specific survival was IJs for S. carpocapsae, and adults for H. bacteriophora. When cadavers were frozen two or three days after infection, few IJs emerged from them. Freezing between five and seven days after infection had no negative effect on IJ production. No decrease in IJ production was measured for H. bacteriophora after freezing. H. bacteriophora also showed improved survival inside versus outside their host when exposed to freezing.     

Gene expression in nematode-infected plant roots

Summary A major pathogen of potato plants (Solanum tuberosum) is the potato cyst nematode (Globodera spp.), which induces localized redifferentiation of a limited number of host cells to form a specialized feeding-site termed the syncytium. A novel strategy utilizing the polymerase chain reaction (PCR) was employed to construct a cDNA library from dissected potato roots highly enriched in syncytial material. The library was differentially screened with cDNA probes derived from the infected root tissue from a compatible interaction and from healthy root tissue. Characterization of one gene identified by the library screen indicated an expression pattern that correlated with events in the immediate vicinity of the pathogen after syncytial establishment. The strategy for library construction and screening could be applicable to the study of gene expression in any plant-pathogen interaction in which the limited supply of cells at the interface of the two organisms precludes a more traditional approach.     

Effects of a novel entomopathogenic nematode-infected host formulation on cadaver integrity, nematode yield, and suppression of Diaprepes abbreviatus and Aethina tumida

An alternative approach to applying entomopathogenic nematodes entails the distribution of nematodes in their infected insect hosts. Protection of the infected host from rupturing, and improving ease of handling, may be necessary to facilitate application. In this study our objective was to test the potential of a new method of formulating the infected hosts, i.e., enclosing the infected host in masking tape. Tenebrio molitor L. cadavers infected with Heterorhabditis indica Poinar, Karunakar and David or Steinernema carpocapsae (Weiser) were wrapped in tape using an automatic packaging machine; the machine was developed to reduce labor and to standardize the final product. The effects of the tape formulation on the ability to protect the cadavers from mechanical damage, nematode yield, and pest control efficacy were tested. After exposure to mechanical agitation at 7-d-post-infection, S. carpocapsae cadavers in tape were more resistant to rupture than cadavers without tape, yet H. indica cadavers 7-d-post-infection were not affected by mechanical agitation (with or without tape), nor was either nematode affected when 4-d-old cadavers were tested. Experiments indicated that infective juvenile yield was not affected by the tape formulation. Laboratory experiments were conducted measuring survival of the root weevil, Diaprepes abbreviatus (L.), or the small hive beetle, Aethina tumida Murray, after the application of two H. indica-infected hosts with or without tape per 15 cm pot (filled with soil). A greenhouse experiment was also conducted in a similar manner measuring survival of D. abbreviatus. In all experiments, both the tape and no-tape treatments caused significant reductions in insect survival relative to the control, and no differences were detected between the nematode treatments. Fifteen days post-application, the infected host treatments caused up to 78% control in A. tumida, 91% control in D. abbreviatus in the lab, and 75% in the greenhouse. These results indicate potential for using the tape-formulation approach for applying nematode infected hosts.     

Formulation and application of the entomopathogenic fungus: Zoophthora radicans (Brefeld) Batko (Zygomycetes: Entomophthorales)

Aims: To isolate and formulate a native strain of Zoophthora radicans naturally infecting larvae of diamondback moth, Plutella xylostella, existing in South Australia and to provide evidence that formulation of the fungus is effective against P. xylostella larvae, and therefore, it could be used as a tool in pest management of this insect. Methods and Results: Dose–response bioassays using formulated and unformulated forms of the fungus strain were carried out against third instar larvae of P. xylostella. Results obtained have indicated a significant increase in the larval mortality when higher concentrations of a formulated form of the fungus strain were applied compared to the treatments with the unformulated form (85·0 vs 57·5% of larval mortality, respectively, at the top concentration of 10⁷ conidia/ml). The median lethal concentration (LC50) for a formulated form was 100 times less than that of the unformulated form when they were applied against the third instar larvae of P. xylostella. In addition, the formulation used in the present bioassays has preserved the viability of introduced fungus conidia for longer time in comparison with the unformulated conidia. Conclusions: The effective application of a formulated fungus strain against P. xylostella larvae constitutes the first step towards its use in pest management of this insect. Significance and Impact of the Study: The formulated fungus in inverted emulsion could be used as an alternative tool to insecticides in pest management of P. xylostella larvae because of the development of resistance to insecticides in the treated larvae.     

Basophil leucocytes in cutaneous hypersensitivity reactions in nematode-infected guinea-pigs.

Cutaneous hypersensitivity reactions were elicited in guinea-pigs infected with the parasitic nematode Trichostrongylus colubriformis by injecting them with a crude extract of T. colubriformis fourth-stage larvae. The reaction was characterized by early oedema and pronounced cellular infiltration, initially with neutrophils but later with mononuclear cells, basophils and variable numbers of eosinophils. Because basophils have been implicated as effector cells in the protective immune response of guinea-pigs to this nematode, the capacity to elicit a basophil-rich cellular infiltrate in infected animals might be a useful assay for T. colubriformis protective antigen(s).     

Antagonism between entomopathogenic fungi and bacterial symbionts of entomopathogenic nematodes

Mutual effects between the symbiotic bacteria of entomopathogenic nematodes, Photorhabdus luminescens and Xenorhabdus poinarii, and entomopathogenic fungi were investigated in vitro. A dual culture assay on nutrient agar supplemented with bromothymol blue and triphenyltetrazolium chloride (NBTA) medium revealed that P. luminescens is antagonistic to Metarhizium anisopliae, Beauveria bassiana, B. brongniartii and Paecilomyces fumosoroseus by inhibiting their growth and conidial production; the fungal growth was not inhibited by X. poinarii. In a second laboratory experiment, crude extract produced by M. anisopliae was tested for its activity against P. luminescens and X. poinarii. Crude extract from M. anisopliae was antibacterial to P. luminescens and X. poinarii at 1000 g/ml and inhibited their growth on NBTA, but had no effect at 100 or 10 g/ml. The influence of the crude extract of M. anisopliae on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis and Steinernema glaseri was assayed on Sabouraud Dextrose Agar (SDA) plates. Results showed that the crude extract of M. anisopliae had no toxic effects even at highest concentration (1000 g/ml).     

Inflammation-induced impairment of enteric nerve function in nematode-infected mice is macrophage dependent

Trichinella spiralis infection in rodents is associated with suppression of ACh release from myenteric plexus that can be mimicked by macrophage-derived cytokines. We verified the presence of a macrophage infiltrate in the intestine during T. spiralis infection and determined the extent to which this cell type is responsible for the neural changes. C57BL/6 mice were infected with 375 T. spiralis larvae by gavage, and the presence of macrophages (F4/80 positive) in the jejunum was determined immunohistochemically. In another experiment, infected mice were treated intravenously with liposomes containing dichloromethylene diphosphonate (clodronate, Cl(2)MDP), which causes apoptosis of macrophages, and killed at postinfection day 6, and jejunal tissues were evaluated for the presence of F4/80-positive cells and for [(3)H]ACh release from the myenteric plexus. Infection caused an infiltration of F4/80-positive cells into the intestinal mucosa, muscle layers, and myenteric plexus region and a significant suppression of ACh release (50%). Depletion of F4/80-positive macrophages using Cl(2)MDP-containing liposomes prevented the suppression in [(3)H]ACh release, identifying macrophages as the cell type involved in the functional impairment of enteric cholinergic nerves.     

Post-application of anti-desiccant agents improves efficacy of entomopathogenic nematodes in formulated host cadavers or aqueous suspension against diapausing codling moth larvae (Lepidoptera: Tortricidae)

Codling moth (CM), Cydia pomonella (L.) is the most serious pest of apple and other pome fruit worldwide. In temperate climates, diapausing cocooned larvae make up 100% of the population. Control of this stage would reduce or eliminate damage by first generation CM in late spring and early summer. Entomopathogenic nematodes (EPNs) are good candidates for control of CM in the cryptic habitats where the larvae overwinter. The two predominant limiting factors for EPNs are adequate moisture and temperatures below 15°C. Formulation that maintains moisture and enables survival of EPN infective juveniles (IJs) until they can infect overwintering larvae would significantly improve their utility for protection of apple, pear and walnut. In laboratory studies conducted in moist mulch (consisting of apple and conifer wood), Galleria mellonella (L.) larvae infected with Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), or Heterorhabditis bacteriophora Poinar and coated with starch and clay, produced mean mortalities of 42, 88, and 24%, respectively in CM larvae. Mulched field plots treated with formulated S. carpocapsae- or S. feltiae-infected G. mellonella larvae, then followed by an application of wood flour foam as an anti-desiccant, resulted in 56 and 86% mortality, respectively. Comparative tests of aqueous suspensions of S. carpocapsae IJs applied to cardboard bands on apple tree trunks followed by water, fire retardant gel or foam resulted in 11, 35, and 85% respective mortalities. Identical tests with S. feltiae resulted in 20, 19, and 97% respective mortalities. Our research with cadaver formulations of EPNs in mulch and aqueous suspensions on tree trunks combined with anti-desiccant agents, demonstrated significant improvement in larvicidal activity for diapausing cocooned CM larvae.     

Suppression of food intake is linked to enteric inflammation in nematode-infected rats

Our aim was to investigate the cause-effect relationship between intestinal inflammation induced by infection with enteric stages of Trichinella spiralis and decreased host food intake. A suppression of food intake in T. spiralis-infected rats occurred within the first 24 h postinfection (PI) and was maximized by day 6 PI. Food intake, cumulated over an 8-day PI period, decreased by 59% compared with uninfected animals. The anti-inflammatory glucocorticoid betamethasone 21-phosphate was orally administered to rats in their drinking water to suppress T. spiralis-induced jejunal inflammation. When treated with a low dose of glucocorticoid (5.2 microg/ml), food intake in infected rats was still significantly reduced, but only by 21% compared with glucocorticoid-treated, uninfected rats. At the highest glucocorticoid dose (10.4 microg/ml) administered, infection-induced reduction in food intake was not different from that of glucocorticoid-treated, uninfected counterparts. The elevation in jejunal myeloperoxidase activity caused by infection was also significantly blunted by oral glucocorticoid treatment. Our results suggest that suppressed host food intake during enteric T. spiralis infection is directly linked to intestinal inflammation.     

The phenotype and function of naturally existing regulatory dendritic cells in nematode-infected mice

Immunosuppression associated with chronic helminth infections has been documented in many studies and regulatory T (Treg) cells have been shown to mediate the nematode-induced immunosuppression, but the role of dendritic cells (DCs) in the induction of Treg cell response and immunosuppression has not yet been fully determined. We analysed the response and function of DCs in mesenteric lymph node (MLNs) of mice infected with a gastrointestinal nematode, Heligmosomoides polygyrus, and observed a substantial expansion of DCs in MLNs following the infection. The CD11c⁺ DCs in MLNs of infected mice showed reduced expression of co-stimulatory molecules CD40, CD86 and MHC-II, and production of inflammatory cytokines IL-12 and IL-6. Analysis of MLN DC subsets defined by CD11c and CD45RB expression showed that the CD11c^lowCD45RB^mid subset increased rapidly following H. polygyrus infection and the CD11c^midCD45RB^high subset expanded from the third week after infection. In the co-culture of sorted DC subsets with ovalbumin-(OVA-)specific T cell receptor (TCR) transgenic CD4⁺ T cells, CD11c^lowCD45RB^mid DCs induced a low proliferation response and a high level of IL-10 production in CD4⁺ T cells, whereas CD11c^midCD45RB^high DCs induced more IFN-γ and IL-4 producing CD4⁺ T cells. Intracellular staining revealed that CD11c^lowCD45RB^mid DCs promoted CD4⁺ Foxp3⁺ differentiations. These results indicate that nematode infections selectively induce expansion of the CD11c^lowCD45RB^mid regulatory DC subset that promotes development of Foxp3⁺ and IL-10 producing Treg cells. The Treg cell responses and immunoregulatory cytokines induced by this regulatory DC subset in turn play an important role in mediation of the nematode-induced immunosuppression.     

Pathogenesis-related genes of entomopathogenic fungi

All living things on Earth experience various diseases such as those caused by viruses, bacteria, and fungi. Insects are no exception to this rule, and fungi that cause disease in insects are called entomopathogenic fungi. These fungi have been developed as microbial insecticides and are used to control various pests. Generally, the mode of action of entomopathogenic fungi is divided into the attachment of conidia, germination, penetration, growth, and generation of secondary infectious conidia. In each of these steps, that entomopathogenic fungi use genes in a complex manner (specific or diverse) has been shown by gene knock-out and RNA-sequencing analysis. In this review, the information mechanism of entomopathogenic fungi was divided into six steps: (1) attachment of conidia to host, (2) germination and appressorium, (3) penetration, (4) fungal growth in hemolymph, (5) conidia production on host, and (6) transmission and dispersal. The strategy used by the fungi in each step was described at the genetic level. In addition, an approach for studying the mode of action of the fungi is presented.     

昆虫病原线虫的耐寒性

昆虫病原线虫可开发成生物农药,广泛应用于多种地下及钻蛀害虫的安全防治。但昆虫病原线虫货架期较短,对寒冷等极端环境的耐受性较差,影响了其在生物防治方面的商业开发。本文介绍了寒区的昆虫病原线虫资源,总结了昆虫病原线虫耐寒性的测定方法及增强方法、耐寒性差异的研究进展,并对其耐寒的生理生化机制及分子机理进行了综述。研究昆虫病原线虫的耐寒性,对于解释种群动态,指导昆虫病原线虫的低温保存,以及拓展其在生物防治方面的应用具有重要意义。     

Formulation of the entomopathogenic fungus Beauveria bassiana JN5R1W1 for the control of mosquito adults and evaluation of its novel applicability

In this study, an effective control agent and control technology for adult mosquitoes using entomopathogenic fungi is proposed as a solution to the adverse effects of chemical insecticides used for mosquito control. A formulation study was conducted using Beauveria bassiana JN5R1W1 with high virulence against both Aedes albopictus and Culex pipiens adult mosquitoes. For the inverted emulsion formulation, three kinds of vegetable oils were used to evaluate the germination promotion, UV blocking effects, and adhesion rate of conidia. As a result, soybean oil was selected as the most effective vegetable oil, and the corresponding formulation was made. Direct contact treatment of the inverted emulsion by spraying resulted in a mortality rate of approximately 100 % for both species of adult mosquitoes. However, in reality, since it is difficult to directly apply fungal agent treatment against adult mosquitoes, we evaluated a control method by indirect contact with mosquitoes after applying the inverted emulsion on a net. As an indirect treatment method, the prepared inverted emulsion showed a mortality rate of approximately 93 % or 89 % against Ae. albopictus and Cx. pipiens adult mosquitoes, respectively. Therefore, these results suggested that the inverted emulsion using B. bassiana JN5R1W1 is an effective control agent for controlling adult mosquitoes.