Increasing the survival and efficacy of entomopathogenic nematodes on exposed surfaces by Pickering emulsion formulations offers new venue for foliar pest management.

Formulation technology has been the primordial focus to improve the low viability and erratic infectivity of entomopathogenic nematodes (EPNs) for foliar application. Adaptability to the fluctuating environment is a key trait in ensuring the survival and efficacy of EPNs. Hence, tailoring formulations towards EPNs foliar applications would effectively deliver consistent and reliable results for above-ground applications. EPNs survival and activity were characterized in novel Pickering emulsion post-application in planta cotton foliage. Two different types of novel formulations, Titanium Pickering emulsion (TPE) and Silica Pickering emulsion Gel (SPEG), were tailored for EPNs foliar applications. We report an extension of survival and infectivity to 96 hrs under controlled conditions by SPEG formulations for survival of IJ's on cotton foliage. In addition, survival of IJs (LT50) was extended from 14hrs in water to > 80 hrs and > 40 hrs by SPEG and TPE respectively. SPEG accounted for the slowest decrease of live IJs per surface area in comparison to TPE and control samples over time, exhibiting a 6-fold increase at 48 hrs. Under extreme conditions, survival and efficacy were extended for 8hrs in SPEG compared to merely 2hrs in control. Potential implications and possible mechanisms of protection are discussed.

Comparative response of Metarhizium brunneum to the cuticles of susceptible and resistant hosts.

Earlier studies demonstrated that Metarhizium brunneum, usually a broad-host pathogen of arthropods, is unable to complete its pathogenic life cycle when inoculated on the fungus-resistant tick, Hyalomma excavatum engorged females. While the fungus penetrates the cuticle of fungus-susceptible tick, Rhipicephalus annulatus females, it is unable to penetrate the cuticle of fungus-resistant tick, and even perishes on its surface. This is probably due to high concentration of antifungal fatty acids and probably also due to a hypersensitive-like response of the tick. To understand the metabolic pathways occurring in the fungal hyphae upon encountering the cuticles, we compared the response of the fungus to cuticle from susceptible and resistant tick cuticles by 2D-gels. The intracellular proteomes of M. brunneum Mb7 exposed to cuticle of the fungus-susceptible tick, R. annulatus, and to the fungus-resistant tick, H. excavatum engorged females were compared after exposure to either cuticles. By means of liquid chromatography-mass spectrometry/mass spectrometry we identified in both proteomes common proteins involved in biological processes as well as unique proteins identified only in the proteome of fungus exposed to fungus-resistant tick cuticle. These proteins were identified in high probability as heat shock proteins, four key enzymes of the glyoxylate cycle, and proteins associated with hypoxia, and exposure to antifungal drugs. These findings are discussed within the M. brunneum-tick pathosystem in relation to tick resistance and host resistance in general.

Can an entomopathogenic nematode serve, as proxy for strongyles, in assessing the anthelmintic effects of phenolic compounds?

As gastro-intestinal nematodes (GINs) become increasingly resistant to chemical anthelmintics, and because consumers scrutinize chemical residues in animal products, the use of herbal anthelmintics and in particular, phenolic compounds, has become attractive. Most life stages of GINs cannot be grown in the lab as they are obligatory parasites, which limits our understanding of the effects of phenolic compounds on their parasitic stages of life. We hypothesized that a species phylogenetically close to GINs and grown in vitro, the insect-parasitic nematode Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade), when fed with Photorhabdus luminescens exposed to plant phenolics, can serve, as proxy for strongyles, in assessing the anthelmintic effects of phenolic compounds. We compared the development of H. bacteriophora infective juveniles (IJ) and the exsheathment rate of L3 larvae of the strongyle Teladorsagia circumcincta and Trichostrongylus colubriformis when exposed to catechin, rutin, chlorogenic and gallic acids, and myricetin. Gallic acid had the highest impact in terms of IJ mortality but the highest impairment of IJ development to adulthood was imposed by myricetin. The studied compounds were not lethal to GINs stricto sensu but we consider that the practical implications of total exsheathment inhibition and mortality on GIN populations are similar. Catechin and rutin had similar effects on rhabditid and strongyles: they imposed ca. 90% lethality of IJs at concentrations higher than 1200 ppm and the remaining live IJs did not develop further, and they also totally inhibited strongyle L3 exsheathment in a dose-response fashion. Gallic acid was 100% lethal to IJs exposed above 300 ppm and chlorogenic acid caused 87% mortality above 1200 ppm, with no development for the surviving IJs but for all lower concentrations, all the IJs developed to adult stages. Likewise, gallic and chlorogenic acids did not affect the exsheatment of GIN L3 larvae. Therefore, a discrepancy between the effects of gallic and chlorogenic acids on the development of rhabditid IJs and exsheathment of GIN L3 larvae was found only when they were exposed to high concentrations. A dose-response of IJ lethality to myricetin was found, with no IJ development between 150 and 2400 ppm; but contrary to the other compounds, myricetin also impaired IJ development of IJs above 10 ppm in a dose-response manner and showed dose-responses in the L3 exsheathment. Apart for the high rates of lethality imposed on IJs by gallic and chlorogenic acids at high concentration, these results suggest that H. bacteriophora fed P. luminescens exposed to phenolics shows potential to serve as model in studies of the anthelmintic effects of phenolics in GIN.

Toxicity of phenolic compounds to entomopathogenic nematodes: A case study with Heterorhabditis bacteriophora exposed to lentisk (Pistacia lentiscus) extracts and their chemical components.

Insects show adaptive plasticity by ingesting plant secondary compounds, such as phenolic compounds, that are noxious to parasites. This work examined whether exposure to phenolic compounds affects the development of insect parasitic nematodes. As a model system for parasitic life cycle, we used Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade) grown with Photorhabdita luminescens supplemented with different concentrations of plant phenolic extracts (0, 600, 1200, 2400 ppm): a crude ethanol extract of lentisk (Pistacia lentiscus) or lentisk extract fractionated along a scale of hydrophobicity with hexane, chloroform and ethyl acetate; and flavonoids (myricetin, catechin), flavanol-glycoside (rutin) or phenolic acids (chlorogenic and gallic acids). Resilience of the nematode to phenolic compounds was stage-dependent, with younger growth stages exhibiting less resilience than older growth stages (i.e., eggs < young juveniles < young hermaphrodites < infective juveniles < mature hermaphrodites). At high concentrations, all of the phenolic compounds studied were lethal to eggs and young juveniles. The nematodes were able to survive in the presence of medium and low concentrations of all studied compounds, but very few of those treatments allowed for reproduction beyond the infective juvenile stage and, at low concentrations, the crude 70% ethanol extract, chloroform and hexane extracts, and myricetin were associated with some impaired reproduction. The ethyl-acetate fraction and gallic acid were extremely lethal to the young stages and allowed almost no development beyond the infective juvenile stage. We conclude that exposure of infective juveniles to phenolics before they infect insects and post-infection exposure of other nematode developmental stages may affect the initiation of the infection, suggesting that the chemistry of dietary phenolics may limit H. bacteriophora's infection of insects.

Ethanolic extracts of Inula viscosa, Salix alba and Quercus calliprinos, negatively affect the development of the entomopathogenic nematode, Heterorhabditis bacteriophora - A model to compare gastro-intestinal nematodes developmental effect.

Heterorhabditis bacteriophora can represent a model system for herbal medication against gastro-intestinal strongylid parasites in determining the recovery and development due to their unique parasitic infectious cycle. The fact that plant extracts impair nematode development is known but their differential impact on stages of the life cycle of H. bacteriophora has never been investigated. We examined the developmental stages resumed from eggs, young juveniles (J1-3), infective juveniles (IJs), young and adult hermaphrodites of H. bacteriophora upon exposure to crude ethanolic extracts of Inula viscosa, Salix alba, and Quercus calliprinos at concentrations of 600, 1200, and 2400ppm. Our results showed that plant extracts were highly toxic to the survival of the eggs and young juveniles J1 to J3 at all concentrations. The plant extracts inhibited their development and were associated with low reproduction parameters (i.e. fecundity and viability of eggs). The IJs, J4, young and developed hermaphrodites displayed concentration-dependent negative effect on development with less egg count, poor vulval muscle development, loss of egg laying capacity and progeny development by matricidal hatching. Plant extract of I. viscosa at low (600ppm) concentration did not impair vulval development. These results suggest that these plant extracts show potential for the control of parasitic rhabditids.

Effects of tannin-rich host plants on the infection and establishment of the entomopathogenic nematode Heterorhabditis bacteriophora.

Parasitized animals can self-medicate. As ingested plant phenolics, mainly tannins, reduce strongyle nematode infections in mammalian herbivores. We investigated the effect of plant extracts known to be anthelmintic in vertebrate herbivores on the recovery of the parasitic entomopathogenic nematode Heterorhabditis bacteriophora infecting African cotton leafworm (Spodoptera littoralis). Nematode infective juveniles (IJs) were exposed to 0, 300, 900, 1200, 2400 ppm of Pistacia lentiscus L. (lentisk), Inula viscosa L. (strong-smelling inula), Quercus calliprinos Decne. (common oak) and Ceratonia siliqua L. (carob) extracts on growth medium (in vitro assay). In control treatments, 50-80% of IJs resumed development to J4, young and developed adult hermaphrodites, whereas all extracts, except for C. siliqua at 300 ppm, impaired IJ exsheathment and development. The highest concentration of I. viscosa extract (2400 ppm) had the strongest effect, killing 95% of exposed nematodes. Surviving nematodes did not recover, remaining at the IJ stage. Over the whole cycle, I. viscosa extract inhibited recovery to 25% or less, and did not allow full development to adulthood, whereas 65% of IJs in the control treatment recovered and resumed development, 12% reaching complete maturation within 72 h of incubation. When herbivorous S. littoralis larvae were fed with different plant extracts in vivo, I. viscosa had the strongest effect at concentrations above 300 ppm, with 90% of insect-invading IJs not developing to parasitic stages, whereas in the control treatment, 85% of IJs resumed development. Exposure to C. siliqua extract also inhibited exsheathment and development of 75% of the IJs. Half of those that resumed development reached full maturation. P. lentiscus and Q. calliprinos extracts also inhibited development of 50% IJs. Our results suggest that H. bacteriophora can be used to study herbal medication against parasites in animals.

Resistant ticks inhibit Metarhizium infection prior to haemocoel invasion by reducing fungal viability on the cuticle surface.

We studied disease progression of, and host responses to, four species in the Metarhizium anisopliae complex expressing green fluorescent protein (GFP). We compared development and determined their relative levels of virulence against two susceptible arthropods, the cattle tick Rhipicephalus annulatus and the lepidopteran Galleria mellonella, and two resistant ticks, Hyalomma excavatum and Rhipicephalus sanguineus. Metarhizium brunneum Ma7 caused the greatest mortality of R. annulatus, Metarhizium robertsii ARSEF 2575 and Metarhizium pingshaense PPRC51 exhibited intermediate levels of virulence, and Metarhizium majus PPRC27 caused low mortality of cattle ticks. Conidia of all four species germinated on all hosts examined, but on resistant hosts, sustained hyphal growth was inhibited and GFP emission steadily and significantly decreased over time, suggesting a loss of fungal viability. Cuticle penetration was observed only for the three most virulent species infecting susceptible hosts. Cuticles of resistant and susceptible engorged female ticks showed significant increases in red autofluorescence at sites immediately under fungal hyphae. This is the first report (i) of tick mortality occurring after cuticle penetration but prior to haemocoel colonization and (ii) that resistant ticks do not support development of Metarhizium germlings on the outer surface of the cuticle. Whether reduced Metarhizium viability on resistant tick cuticles is due to antibiosis or limited nutrient availability is unknown.

A realistic appraisal of methods to enhance desiccation tolerance of entomopathogenic nematodes.

Understanding the desiccation survival attributes of infective juveniles of entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis, is central to evaluating the reality of enhancing the shelf-life and field persistence of commercial formulations. Early work on the structural and physiological aspects of desiccation survival focused on the role of the molted cuticle in controlling the rate of water loss and the importance of energy reserves, particularly neutral lipids. The accumulation of trehalose was also found to enhance desiccation survival. Isolation of natural populations that can survive harsh environments, such as deserts, indicated that some populations have enhanced abilities to survive desiccation. However, survival abilities of EPN are limited compared with those of some species of plant-parasitic nematodes inhabiting aerial parts of plants. Research on EPN stress tolerance has expanded on two main lines: i) to select strains of species, currently in use commercially, which have increased tolerance to environmental extremes; and ii) to utilize molecular information, including expressed sequence tags and genome sequence data, to determine the underlying genetic factors that control longevity and stress tolerance of EPN. However, given the inherent limitations of EPN survival ability, it is likely that improved formulation will be the major factor to enhance EPN longevity and, perhaps, increase the range of applications.

Survival and efficacy of entomopathogenic nematodes on exposed surfaces.

Entomopathogenic nematodes (EPN) species differ in their capability to withstand rapid desiccation (RD). Infective juveniles of Steinernema carpocapsae are a better adaptable and tolerant than Steinernema feltiae or Heterorhabditis bacteriophora as, an optimal RH of > 90% is required by S. feltiae and H. bacteriophora while maintaining RH equivalent to 74% could sustain survival of S. carpocapsae under RD. Our findings from infectivity suggest that following application, shrunk IJs are acquired passively by the larvae, probably rehydrate and resume infection within the insect gut. Water loss rate is a key factor affecting survival of S. carpocapsae on exposed surfaces. The present study provides the foundation for characterizing mechanism of rapid rate of water loss in EPN. ATR-FTIR is a rapid and reliable method for analysis of water loss. Changes in peak intensity was observed at 3100-3600 cm-1 (OH bonds of water), 2854 cm-1 (CH stretching of symmetric CH2, acyl chains), 2924 cm-1 (CH stretching of anti-symmetric CH2, lipid packing heterogeneity), 1634 cm-1 (amide I bonds) indicate major regions for hydration dependent changes in all EPN species. FTIR data also indicates that, S. carpocapsae contains strong water interacting regions in their biochemical profile, which could be an influencing factor in their water holding capacity under RD. ATR-FTIR were correlated to water content determined gravimetrically by using Partial Least square -Regression and FTIR multivariate method, which could be used to screen a formulation's potential to maintain or delay the rate of water loss in a rapid and efficient manner.

Individual Coating of Entomopathogenic Nematodes with Titania (TiO2) Nanoparticles Based on Oil-in-Water Pickering Emulsion: A New Formulation for Biopesticides.

This study presents a new eco-friendly formulation of entomopathogenic nematodes (EPNs) based on individual coating of EPNs with titanium dioxide (TiO2) nanoparticles (NPs) and mineral oil via oil-in-water Pickering emulsions. Mineral oil-in-water emulsions stabilized by amine-functionalized titanium dioxide (TiO2-NH2) particles were prepared. 40:60 and 50:50 oil-water volume ratios using 2 wt % TiO2-NH2 particles were found to be the most stable emulsions with a droplet size suitable for the formulation and were further studied for their toxicity against the incorporated EPNs. Carboxyfluorescein was covalently bonded to TiO2-NH2 NPs, and the resulting composite was observed via fluorescence confocal microscopy. The dry coating was evaluated using SEM and confocal microscopy, which showed significant nematode coverage by the particles and oil. The final formulation was biocompatible with the studied EPNs, where the viability of the EPNs in the formulation was equivalent to control aqueous suspension after 120 days. Finally, yields of nematodes from infected Galleria mellonella cadavers collected for 150 days showed no significant differences (P > 0.05) using the tested emulsions compared to the control containing nematodes in water.

Behavioral and molecular response of the insect parasitic nematode Steinernema carpocapsae to cues emitted by a host, the red palm weevil, Rhynchophorus ferrugineus.

As the larvae of the date palm pest, the red palm weevil, Rhynchophorus ferrugineus, feeds on the host tissue, they emit a distinctive sound which can be recorded outside of the infected tree. We evaluated the response of infective juveniles (IJs) of the entomopathogenic nematodes Steinernema carpocapsae to the R. ferrugineus larvae and it's sound source, separately. In the presence of the insect larvae, 50.2 % of total IJs moved toward those larvae. Recorded insect larvae sound emitted by the speaker resulted in 7% of total IJs near the sound source. RNA-Seq data indicated that more genes were downregulated in S. carpocapsae IJs exposed to insect and speaker compared to non-stimulated IJs. IJs exposed to insect exhibited more up-regulated genes than IJs exposed to speaker. Enriched pathways and biological processes in IJs were similar for both stimuli. The inhibition of locomotion, regulation of neurotransmitter secretion, response to biotic stimulus, and cellular response to chemical stimuli were enriched with unique GO terms for speaker treatment. The regulation of localization, sodium ion transmembrane transport, regulation of response to stress and response to organic substances were the GO categories enriched unique to insect. The host-parasitic interaction was regulated by the differential expression of Ras/MAP kinase, TGF-beta signaling, insulin signaling, AMPK signaling, PPAR signaling pathways and many developmental pathways. More prominent R. ferrugineus host localization by S. carpocapsae was primarily due to the differential transcriptional regulation of olfactory signal transduction, FOXO-family proteins, calcium signaling, WNT and mTOR signaling pathway. The neural basis for the nematode attraction to insect host is based on the chemosensation and the mechanosensation. Many neuropeptides and neuromodulators are involved in regulating the foraging behavior of S. carpocapsae. The results of this study provide new insights into the molecular mechanisms that allow these nematodes to seek insect hosts. Our finding, especially the molecular ones suggest that chemical cues emitted by the active insect host are stimulants of nematodes attraction. Whereas the sound emitted by the insect has minor effects on the nematode behavior.

Novel technique for quantifying adhesion of Metarhizium anisopliae conidia to the tick cuticle.

The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of Metarhizium anisopliae var. anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged Rhipicephalus annulatus (Say) (Arachnida: Ixodidae) females and was also verified for M. anisopliae var. acridum Driver et Milner (Hypocreales: Clavicipitaceae) and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female R. annulatus and tick mortality. However, no correlation was observed between a tick's susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed.

Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface.

Conidial germination and the formation of appressoria are important events in the interactions between entomopathogenic fungi and their arthropod hosts. In this study, we demonstrate the effects of lipids extracted from tick epicuticle and the surface of a mammalian host (calf) on conidial germination and the development of appressoria in two subspecies of Metarhizium anisopliae, M. anisopliae var. anisopliae (M.an.an.-7) and M. anisopliae var. acridum (M.an.ac.-5), which have different levels of virulence toward ticks. Pentane extracts of epicuticles of ticks susceptible and resistant to fungal infection always stimulated the germination of M.an.an.-7 conidia and the development of their appressoria; whereas the effects of dichloromethane (DCM) extracts of tick epicuticle varied depending on the tick. The DCM extracts from most of the tick species and developmental stages stimulated conidial germination and/or the formation of appressoria in M.an.an.-7. However, a DCM extract of lipids from the most resistant tick, engorged Hyalomma excavatum female, inhibited the germination of M.an.an.-7 conidia. Conidia of the non-virulent M.an.ac.-5 did not germinate on agarose amended with any of the examined tick extracts. However, when the tick extracts were placed on bactoagar, conidial germination increased 7- to 8-fold. Extracts from the skin, hair and ear secretions of a calf stimulated conidial germination and the formation of appressoria in M.an.an.-7, but not M.an.ac.-5. This study demonstrates that lipids from tick epicuticles and mammalian skin selectively affect the germination of conidia of entomopathogenic fungi. The effects of these lipids may explain the variability in tick control these fungi provide for different hosts.

Characterization of the phenotypic and genotypic tolerance to abiotic stresses of natural populations of Heterorhabditis bacteriophora.

Entomopathogenic nematodes are effective biocontrol agents against arthropod pests. However, their efficacy is limited due to sensitivity to environmental extremes. The objective of the present study was to establish a foundation of genetic-based selection tools for beneficial traits of heat and desiccation tolerance in entomopathogenic nematodes. Screening of natural populations enabled us to create a diverse genetic and phenotypic pool. Gene expression patterns and genomic variation were studied in natural isolates. Heterorhabditis isolates were phenotyped by heat- and desiccation-stress bioassays to determine their survival rates compared to a commercial line. Transcriptomic study was carried out for the commercial line, a high heat-tolerant strain, and for the natural, low heat-tolerant isolate. The results revealed a higher number of upregulated vs. downregulated transcripts in both isolates vs. their respective controls. Functional annotation of the differentially expressed transcripts revealed several known stress-related genes and pathways uniquely expressed. Genome sequencing of isolates with varied degrees of stress tolerance indicated variation among the isolates regardless of their phenotypic characterization. The obtained data lays the groundwork for future studies aimed at identifying genes and molecular markers as genetic selection tools for enhancement of entomopathogenic nematodes ability to withstand environmental stress conditions.

Pathogenicity of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) to tick eggs and the effect of egg cuticular lipids on conidia development.

The ovicidal efficacy of two entomopathogenic hyphomycetes fungi--Metarhizium anisopliae variety acridum (M. an. ac.) Driver and Milner (Hypocreales: Clavicipitaceae) and Metarhizium anisopliae variety anisopliae (M. an. an.) (Metschn.) Sorokin (Hypocreales: Clavicipitaceae)--was evaluated against eggs of three tick species (Acari: Ixodidae)--Hyalomma excavatum (Koch), Rhipicephalus (Boophilus) annulatus (Say), and Rhipicephalus sanguineus (Latereille)--by placing eggs, laid by surface-sterilized females, on conidia-impregnated filter paper. Although M. an. an. strains differed in their virulence to the tested ticks, they reduced the hatching percentages of eggs of all three tick species to 0-32% compared with 80-90% in the control eggs. The M. an. ac. strains were found highly virulent to H. excavatum and R. sanguineus eggs, reducing the hatching percentages to 2-6% but had no influence on hatching of R. annulatus eggs. Older tick eggs were more susceptible to fungal infection than newly laid ones. The effects of polar and nonpolar lipid fractions, extracted from the surface of tick eggs, on the development of conidia were tested. Both germination of M. an. an. conidia and formation of appressoria were stimulated by extracts from egg cuticles of all three tested tick species. However, the stimulating effect was lower when the conidia were exposed to lipids from relatively less susceptible R. annulatus eggs than when exposed to lipids from H. excavatum or R. sanguineus eggs. Unlike those of M. an. an., conidia of M. an. ac. exposed to such lipid extracts did not germinate and did not form appressoria.

Expression of different desiccation-tolerance related genes in various species of entomopathogenic nematodes.

Entomopathogenic nematodes used as biological control agents encounter various stress conditions during extended periods in the soil. We investigated gene expression in nematodes that were tolerant or susceptible to desiccation stress to determine whether enhanced tolerance in these populations results from a 'gene-expression response' to desiccation or if, for enhanced tolerance, no such response is needed, perhaps due to a state of constant 'readiness'. The expressions of four genes, aldehyde dehydrogenase, nucleosome assembly protein 1, glutathione peroxidase and heat-shock protein 40, were characterized during desiccation stress in five entomopathogenic nematode species with differing stress tolerance: Steinernema feltiae strain IS-6, S. feltiae Carmiel strain, Steinernema carpocapsae Mexican strain, Steinernema riobrave, and Heterorhabditis bacteriophora strain TTO1. After 24h of desiccation, we observed an inverse relationship between expression of the studied genes and phenotypic desiccation-tolerance capability in the nematodes. H. bacteriophora TTO1 was most susceptible to desiccation but showed the highest expression of all studied genes under desiccation. S. carpocapsae Mexican strain and S. riobrave showed the lowest expression of these genes but were most tolerant to desiccation. Our study showed no induction of gene expression in stress-tolerant nematodes, whereas the stress-susceptible nematodes responded to stress by induced expression of these genes. Since the different levels of gene expression were found to be related to the different stress-tolerance capabilities of the nematodes, these gene-expression ratios can potentially be used as markers of desiccation tolerance in entomopathogenic nematodes.

Proteomic analysis of the entomopathogenic nematode Steinernema feltiae IS-6 IJs under evaporative and osmotic stresses.

In order to improve the storage capability under desiccation of the widely sold biological insecticides based on entomopathogenic nematodes (EPNs), we need to understand how these organisms respond to desiccation stress. As part of our studies to achieve this, we studied survival and protein expression in infective juveniles of the EPN Steinernema feltiae IS-6 when exposed to evaporative (exposure to 97% relative humidity (RH) for 3 days, followed by a 1-day exposure to 85% RH) and osmotic (exposure to 24% glycerol for 8h) stresses. More than 400 protein spots that were detected by proteomic analysis showed reproducible abundance within replications. Of these, 10 spots and 7 spots showed detectable changes in abundance under evaporative and osmotic stress, respectively, compared to fully hydrated nematodes. Three spots exhibited a differential response pattern between evaporative and osmotic desiccation (one was down regulated and two were novel in evaporative desiccation). Peptide mass mapping with MALDI-TOF mass spectrometry (MS) identified 10 desiccation-response proteins, among which several are known to be stress responsive including heat shock protein 60, coenzyme q biosynthesis protein, inositol monophosphatase and fumarate lyase that were found in both stresses. Other identified proteins are known to be involved in the cell cycle regulation, regulation of gene transcription, organization of macromolecular structure and some currently have no known functions. Our results suggest that it is unlikely that improvement of desiccation tolerance in EPNs can be achieved through genetic transformation and addition of single genes and that selective breeding could be the best approach to generate desiccation resistant worms.

Transcriptome analysis of stress tolerance in entomopathogenic nematodes of the genus Steinernema.

Entomopathogenic nematodes of the genus Steinernema are effective biological control agents. The infective stage of these parasites can withstand environmental stresses such as desiccation and heat, but the molecular and physiological mechanisms involved in this tolerance are poorly understood. We used 454 pyrosequencing to analyse transcriptome expression in Steinernema spp. that differ in their tolerance to stress. We compared these species, following heat and desiccation treatments, with their non-stressed counterparts. More than 98% of the transcripts found matched homologous sequences in the UniRef90 database, mostly nematode genes (85%). Among those, 60.8% aligned to the vertebrate parasites including Ascaris suum, Loa loa, and Brugia malayi, 23.3% aligned to bacteriovores, mostly from the genus Caenorhabditis, and 1% aligned to EPNs. Analysing gene expression patterns of the stress response showed a large fraction of down-regulated genes in the desiccation-tolerant nematode Steinernema riobrave, whereas a larger fraction of the genes in the susceptible Steinernema feltiae Carmiel and Gvulot strains were up-regulated. We further compared metabolic pathways and the expression of specific stress-related genes. In the more tolerant nematode, more genes were down-regulated whereas in the less tolerant strains, more genes were up-regulated. This phenomenon warrants further exploration of the mechanism governing induction of the down-regulation process. The present study revealed many genes and metabolic cycles that are differentially expressed in the stressed nematodes. Some of those are well known in other nematodes or anhydrobiotic organisms, but several are new and should be further investigated for their involvement in desiccation and heat tolerance. Our data establish a foundation for further exploration of stress tolerance in entomopathogenic nematodes and, in the long term, for improving their ability to withstand suboptimal environmental conditions.

Stressed worms: responding to the post-genomics era.

Nematodes are among the most successful organisms in withstanding stress conditions associated with water loss, and viable individuals have been recovered from dry desert soils. Little is known about the biochemical and molecular events underpinning nematodes' physiological responses to dehydration. Post-genomics research in Caenorhabditis elegans may offer an opportunity to understand the stress response better. This review focuses on recent progress in understanding the molecular mechanisms of water-loss associated stress response in the model nematode C. elegans and in parasitic nematodes and discusses the scope for applying the knowledge and tools derived from a model organism for the study of wild, environmentally-adapted, parasitic nematodes, in the light of the emergence of genomics research of non-model organisms.

An LEA group 3 family member is involved in survival of C. elegans during exposure to stress.

In order to establish a functional role for late embryogenesis abundant (LEA) proteins in response to stress conditions in Caenorhabditis elegans, we silenced the expression of an LEA (Ce-lea-1) gene and determined the survival of worms under stress conditions. Ce-lea-1 transcription was induced during dehydration of C. elegans dauer juveniles. Following partial silencing of Ce-lea-1 transcription, we demonstrated a specific and significant reduction in worm survival during induction of desiccation, osmotic and heat stress. Together, these results establish a functional role for Ce-lea-1 in stress survival of C. elegans and suggest that Ce-lea-1 may function as a component that is common to the responses to the examined stress conditions.